Nucleic acids encoding FGF21-Fc fusion proteins

ABSTRACT

The invention relates to nucleic acids encoding fusion proteins comprising polypeptide and protein variants of fibroblast growth factor 21 (FGF21) with improved pharmaceutical properties. Also disclosed are vectors and host cells comprising the same and methods of producing the fusion proteins.

This application is a continuation of U.S. patent application Ser. No.14/987,338, filed Jan. 4, 2016, which is a divisional application ofU.S. patent application Ser. No. 14/630,206, filed Feb. 24, 2015, nowU.S. Pat. No. 9,266,935, which is a divisional of U.S. patentapplication Ser. No. 13/626,194, filed Sep. 25, 2012, now U.S. Pat. No.9,006,400, which claims priority to U.S. Provisional Application No.61/539,280, filed Sep. 26, 2011, all of which are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to new fusion proteins comprisingfibroblast growth factor 21 (FGF21) known to improve metabolic profilesin subjects to whom they are administered.

BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family is characterized by 22genetically distinct, homologous ligands, which are grouped into sevensubfamilies. FGF-21 is most closely related to, and forms a subfamilywith, FGF-19 and FGF-23. This FGF subfamily regulates diversephysiological processes uncommon to classical FGFs, namely energy andbile acid homeostasis, glucose and lipid metabolism, and phosphate aswell as vitamin D homeostasis. Moreover, unlike other FGFs, thissubfamily acts in an endocrine fashion. (Moore, D. D. (2007) Science316, 1436-8)(Beenken et al. (2009) Nature Reviews Drug Discovery 8,235).

FGF21 is a 209 amino acid polypeptide containing a 28 amino acid leadersequence (SEQ ID NO:5). Human FGF21 has about 79% amino acid identity tomouse FGF21 and about 80% amino acid identity to rat FGF21. Fibroblastgrowth factor 21 (FGF21) has been described as a treatment for ischemicvascular disease, wound healing, and diseases associated with loss ofpulmonary, bronchia or alveolar cell function. (Nishimura et al. (2000)Biochimica et Biophysica Acta, 1492:203-206; patent publicationWO01/36640; and patent publication WO01/18172) Although FGF-21 activatesFGF receptors and downstream signaling molecules, including FRS2a andERK, direct interaction of FGFRs and FGF-21 has not been detected.Studies have identified β-klotho, which is highly expressed in liver,adipocytes and pancreas, as a determinant of the cellular response toFGF-21 and a cofactor which mediates FGF-21 signaling through FGFRs(Kurosu, H. et al. (2007) J Biol Chem 282, 26687-95). FGF21 is a potentagonist of the FGFR1(IIIc), FGFR2(IIIc) and FGFR3(IIIc) β-klothosignaling complexes.

FGF-21 has been shown to induce insulin-independent glucose uptake.FGF-21 has also been shown to ameliorate hyperglycemia in a range ofdiabetic rodent models. In addition, transgenic mice over-expressingFGF-21 were found to be resistant to diet-induced metabolicabnormalities, and demonstrated decreased body weight and fat mass, andenhancements in insulin sensitivity (Badman, M. K. et al. (2007) CellMetab 5, 426-37). Administration of FGF-21 to diabetic non-humanprimates caused a decline in fasting plasma glucose, triglycerides,insulin and glucagon levels, and led to significant improvements inlipoprotein profiles including a nearly 80% increase in HDL cholesterol(Kharitonenkov, A. et al. (2007) Endocrinology 148, 774-81). Recentstudies investigating the molecular mechanisms of FGF21 action haveidentified FGF21 as an important endocrine hormone that helps to controladaptation to the fasting state. (Badman et al. (2009) Endocrinology150, 4931)(Inagaki et al. (2007) Cell Metabolism 5, 415) This provides apreviously missing link downstream of PPARα, by which the livercommunicates with the rest of the body in regulating the biology ofenergy homeostasis. (Galman et al. (2008) Cell Metabolism 8,169)(Lundasen et al. (2007) Biochemical and Biophysical ResearchCommunications 360, 437).

FGF21 regulates adipocyte homeostasis through activation of anAMPK/SIRT1/PGC1α pathway to inhibit PPARγ expression and increasemitochondrial function. (Chau et al. (2010) PNAS 107, 12553) FGF21 alsoincreases glucose uptake by skeletal muscle as measured in culturedhuman myotubes and isolated mouse tissue. FGF21 treatment of rodentislet cells leads to improved function and survival through activationof ERK½ and Akt pathways. (Wente et al. (2006) Diabetes 55, 2470) FGF21treatment also results in altered gene expression for lipogenesis andfatty acid oxidation enzymes in rodent livers, likely through HNF4α andFoxa2 signaling.

A difficulty associated with using FGF-21 directly as a biotherapeuticis that its half-life is very short. (Kharitonenkov, A. et al. (2005)Journal of Clinical Investigation 115:1627-1635) In mice, the half-lifeof human FGF21 is 0.5 to 1 hours, and in cynomolgus monkeys, thehalf-life is 2 to 3 hours. FGF21 may be utilized as a multi-use, sterilepharmaceutical formulation. However, it has been determined thatpreservatives, i.e., m-cresol, have an adverse affect on its stabilityunder these conditions.

In developing an FGF21 protein for use as a therapeutic in the treatmentof type 1 and type 2 diabetes mellitus and other metabolic conditions,an increase in half-life and stability would be desirable. FGF21proteins having enhanced half-life and stability would allow for lessfrequent dosing of patients being administered the protein. Clearly,there is a need to develop a stable aqueous protein formulation for thetherapeutic protein FGF21.

Furthermore, significant challenge in the development of FGF21 as aprotein pharmaceuticals, is to cope with its physical and chemicalinstabilities. The compositional variety and characteristics of proteinsdefine specific behaviors such as folding, conformational stability, andunfolding/denaturation. Such characteristics should be addressed whenaiming to stabilize proteins in the course of developing pharmaceuticalformulation conditions utilizing aqueous protein solutions (Wang, W.,Int. J. of Pharmaceutics, 18, (1999)). A desired effect of stabilizingtherapeutic proteins of interest, e.g., the proteins of the presentinvention, is increasing resistance to proteolysis and enzymaticdegradation, thereby improving protein stability and reducing proteinaggregation.

SUMMARY OF THE INVENTION

The invention relates to the identification of new fusion proteins whichcomprise fibroblast growth factor 21 (FGF21) and which have improvedpharmaceutical properties over the wild-type FGF21 and variants thereofunder pharmaceutical formulation conditions, e.g., are more stable,possess the ability to improve metabolic parameters for subjects to whomthey are administered, are less susceptible to proteolysis and enzymaticdegradation, and are less likely to aggregate and form complexes. Thefusion proteins of the invention comprise truncations, mutations, andvariants of FGF21.

Also disclosed are methods for treating FGF21-associated disorders, aswell as other metabolic, endocrine, and cardiovascular disorders, suchas obesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, acute myocardialinfarction, hypertension, cardiovascular disease, atherosclerosis,peripheral arterial disease, stroke, heart failure, coronary heartdisease, kidney disease, diabetic complications, neuropathy,gastroparesis, disorders associated with severe inactivating mutationsin the insulin receptor, and other metabolic disorders, and in reducingthe mortality and morbidity of critically ill patients.

The fusion proteins of the present invention may be used as a onceweekly injectable either alone or in combination with oral anti-diabeticagents which will improve the glycemic control, body weight and lipidprofile of type 1 and type 2 diabetes mellitus patients. The proteinsmay also be used for the treatment of obesity or other FGF21-associatedconditions.

The fusion proteins of the invention overcome the significant hurdles ofphysical instabilities associated with protein therapeutics, including,for instance, with the administration of the wild-type FGF21, bypresenting proteins which are more stable, less susceptible toproteolysis and enzymatic degradation, and less likely to aggregate andform complexes, than wild-type FGF21 under pharmaceutical formulationconditions.

In a first aspect, the invention provides Fibroblast Growth Factor 21(FGF21) fusion proteins comprising one or more of the sequences listedin Table 1, and further described herein. The FGF21 sequences listed inTable 1 may be variants of the wild-type FGF21 sequence, e.g., thewild-type FGF21 sequence with NCBI reference number NP_061986.1, andfound in such issued patents as, e.g., U.S. Pat. No. 6,716,626B1,assigned to Chiron Corporation.

Said fusions may be, for example, between the variant FGF21 sequences,e.g., the sequences of Table 1, and other molecules (a non-FGF21portion), e.g., an IgG constant domain or fragment thereof (e.g., the Fcregion), Human Serum Albumin (HSA), or albumin-binding polypeptides. Ina preferred embodiment, the non-FGF21 portion of the molecule is an Fcregion.

Other embodiments are drawn to polynucleotides encoding the fusionproteins of the invention, a vector containing said polynucleotides anda host cell carrying said vector.

Provided herein are methods used to generate the fusion proteins of theinvention, wherein such methods involve modification of the wild-typeFGF21 protein, via e.g., the site-specific incorporation of amino acidsat positions of interest within the wild-type FGF21 protein, as well asthe fusion between the FGF21 portion of the molecule to other molecules,e.g., an IgG constant domain or fragment thereof (e.g., the Fc region),Human Serum Albumin (HSA), or albumin-binding polypeptides. Saidmodifications and fusions enhance the biological properties of thefusion proteins of the invention relative to the wild-type versions ofthe proteins as well as, in some cases, serving as points of attachmentfor, e.g., labels and protein half-life extension agents, and forpurposes of affixing said variants to the surface of a solid support.Related embodiments of the invention are methods to produce cellscapable of producing said proteins of the invention, and of producingvectors containing DNA encoding said variants and fusions.

In various embodiments, the fusion proteins of the invention disclosedherein can comprise one or more fragments of the FGF21 wild-typesequences, including fragments as small as 8-12 amino acid residues inlength, and wherein the polypeptide is capable of lowering blood glucosein a mammal. In various embodiments, the fusion proteins of theinvention disclosed herein can comprise one or more variant of the FGF21wild-type sequences, e.g., with one or more amino acid deletion,insertion, addition, or substitution relative to the wild-type sequencesthereof.

In some embodiments, the fusion proteins of the invention disclosedherein can be covalently linked to one or more polymers, such aspolyethylene glycol (PEG) or polysialic acid, whether at the position ofsite-specific amino acid modifications made relative to the wild-typeFGF21, or at the position of amino acids commonly shared with thewild-type versions of those proteins. The PEG group is attached in sucha way so as enhance, and/or not to interfere with, the biologicalfunction of the constituent portions of the fusion proteins of theinvention, e.g., the FGF21 protein variants. In other embodiments, thepolypeptides of the invention can be fused to a heterologous amino acidsequence, optionally via a linker, such as GS, GGGGSGGGGSGGGGS (SEQ IDNO:6). The heterologous amino acid sequence can be an IgG constantdomain or fragment thereof (e.g., the Fc region), Human Serum Albumin(HSA), or albumin-binding polypeptides. Such fusion proteins disclosedherein can also form multimers.

In some embodiments, a heterologous amino acid sequence (e.g., HSA, Fc,etc.) is fused to the amino-terminal of the fusion proteins of theinvention. In other embodiments, the fusion heterologous amino acidsequence (e.g., HSA, Fc, etc.) is fused to the carboxyl-terminal of thefusion proteins of the invention.

Yet another embodiment is drawn to methods of treating a patientexhibiting one or more FGF21-associated disorders, such as obesity, type2 diabetes mellitus, type 1 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, acute myocardialinfarction, hypertension, cardiovascular disease, atherosclerosis,peripheral arterial disease, stroke, heart failure, coronary heartdisease, kidney disease, diabetic complications, neuropathy,gastroparesis, disorders associated with inactivating mutations in theinsulin receptor, and other metabolic disorders, comprisingadministering to said patient in need of such treatment atherapeutically effective amount of one or more proteins of theinvention or a pharmaceutical composition thereof.

The invention also provides pharmaceutical compositions comprising thefusion proteins of the invention disclosed herein and a pharmaceuticallyacceptable formulation agent. Such pharmaceutical compositions can beused in a method for treating a metabolic disorder, and the methodcomprises administering to a human patient in need thereof apharmaceutical composition of the invention. Non-limiting examples ofmetabolic disorders that can be treated include type 1 and type 2diabetes mellitus and obesity.

These and other aspects of the invention will be elucidated in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show V188 has improved efficacy in the ob/ob diabetic mousemodel over V76. V188 shows superior results when administered at 1milligram per kilogram (mpk), compared to the 5 milligram per kilogramat which V76 was administered. FIG. 1A shows fed plasma glucose as areadout (circles represent vehicle (PBS—phosphate buffered saline),squares represent V76 at 5 mpk, and triangles represent V188 at 1 mpk.FIG. 1B shows fed plasma insulin as a readout (from left to right:vehicle, V76 at 5 mpk, and V188 at 1 mpk). FIG. 1C shows body weight asa readout (from left to right: vehicle, V76 at 5 mpk, and V188 at 1mpk). FIG. 1D shows liver lipid content as a readout (from left toright: vehicle, V76 at 5 mpk, and V188 at 1 mpk).

FIGS. 2A-2D show V101 has improved efficacy in the ob/ob diabetic mousemodel over V76. V101 shows superior results when administered at 1milligram per kilogram (mpk), compared to the 5 milligram per kilogramat which V76 was administered. FIG. 2A shows fed plasma glucose as areadout (circles represent vehicle (PBS—phosphate buffered saline),squares represent V76 at 5 mpk, and triangles represent V101 at 1 mpk.FIG. 2B shows fed plasma insulin as a readout (from left to right:vehicle, V76 at 5 mpk, and V101 at 1 mpk). FIG. 2C shows body weight asa readout (from left to right: vehicle, V76 at 5 mpk, and V101 at 1mpk). FIG. 2D shows liver lipid content as a readout (from left toright: vehicle, V76 at 5 mpk, and V101 at 1 mpk).

FIGS. 3A-3D show V103 has improved efficacy in the ob/ob diabetic mousemodel over V76. V103 shows superior results when administered at 1milligram per kilogram (mpk), compared to the 5 milligram per kilogramat which V76 was administered. FIG. 3A shows fed plasma glucose as areadout (circles represent vehicle (PBS—phosphate buffered saline),squares represent V76 at 5 mpk, and triangles represent V103 at 1 mpk.FIG. 3B shows fed plasma insulin as a readout (from left to right:vehicle, V76 at 5 mpk, and V103 at 1 mpk). FIG. 3C shows body weight asa readout (from left to right: vehicle, V76 at 5 mpk, and V103 at 1mpk). FIG. 3D shows liver lipid content as a readout (from left toright: vehicle, V76 at 5 mpk, and V103 at 1 mpk).

FIGS. 4A-4D demonstrate the superior pharmacokinetic and thermocynamicproperties possessed by the fusion proteins of the invention over FGF21fusion proteins in the art. FIG. 4A shows the plasma concentrations offusion proteins of the invention in PCT Publication WO10/129600described as Fc-L (15)-FGF21 (L98R, P171G) and Fc-L (15)-FGF21 (L98R,P171G, A180E), following the IV injection of said fusion in mice. FIG.4B shows pharmacokinetic properties of the fusion proteins of theinvention (V101, V103 & V188) after a single IV dose in the mouse asassayed by anti-Fc-ELISA compared with pharmacokinetic data generated inthe mouse for V76 in a previous study using an anti-FGF21 antibodyELISA. FIG. 4C shows a spot check of the fusion proteins of theinvention in an anti-FGF21 Western blot, consistent with anti-Fc-ELISAdata at 120 hours and 15 days. The samples in the blot are as follows: Arepresents V101, B represents V103, and C represents V188. Control isV101 and serum. FIG. 4D demonstrates the significantly increasedthermodynamic stability of the fusion proteins of the invention comparedto V76. From top to bottom, the figure represents V101, V103, and V188,all of which have improved melting temperatures (T_(m)) compared to V76(T_(m)<50° C. (not shown)) and wild-type FGF21 (T_(m)=46.5° C.±0.3 (notshown)).

DETAILED DESCRIPTION OF THE INVENTION

The fusion proteins of the present invention represent modified versionsof the full length, wild-type FGF21 polypeptide, as known in the art.FGF21 wild-type sequence will serve as a reference sequence (SEQ IDNO:1), for instance, when comparisons between the FGF21 wild-typesequence and the protein variants are necessary. The FGF21 wild-typesequence has NCBI reference sequence number NP_061986.1, and can befound in such issued patents as, e.g., U.S. Pat. No. 6,716,626B1,assigned to Chiron Corporation (SEQ ID NO:1).

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1               5                  10                  15Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro            20                  25                  30Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr        35                  40                  45Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg    50                  55                  60Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu65                  70                  75                  80Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                85                  90                  95Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly            100                 105                 110Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu        115                 120                 125Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu    130                 135                 140His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly145                 150                 155                 160Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu                165                 170                 175Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp            180                 185                 190Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala        195                 200                 205 Ser 209

The corresponding mRNA sequence coding for the full-length FGF21polypeptide (NCBI reference sequence number NM_019113.2) is shown below(SEQ ID NO:2)

1 ctgtcagctg aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc 61acctggacaa ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc 121ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg gttcgagcac 181tcaggactgt gggtttctgt gctggctggt cttctgctgg gagcctgcca ggcacacccc 241atccctgact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 301acagatgatg cccagcagac agaagcccac ctggagatca gggaggatgg gacggtgggg 361ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 421attcaaatct tgggagtcaa gacatccagg ttcctgtgcc agcggccaga tggggccctg 481tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 541ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 601tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 661ccccccgcac tcccggagcc acccggaatc ctggcccccc agccccccga tgtgggctcc 721tcggaccctc tgagcatggt gggaccttcc cagggccgaa gccccagcta cgcttcctga 781agccagaggc tgtttactat gacatctcct ctttatttat taggttattt atcttattta 841tttttttatt tttcttactt gagataataa agagttccag aggagaaaaa aaaaaaaaaa 901aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

The mature FGF21 sequence lacks a leader sequence and may also includeother modifications of a polypeptide such as proteolytic processing ofthe amino terminus (with or without a leader sequence) and/or thecarboxyl terminus, cleavage of a smaller polypeptide from a largerprecursor, N-linked and/or O-linked glycosylation, and otherpost-translational modifications understood by those with skill in theart. A representative example of a mature FGF21 sequence has thefollowing sequence (SEQ ID NO:3, which represents amino acid positions29-209 of full length FGF21 protein sequence (NCBI reference sequencenumber NP_061986.1)):

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val                 5                  10                  15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His             20                  25                  30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser         35                  40                  45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln     50                  55                  60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65                   70                  75                  80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg                 85                  90                  95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His            100                 105                 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro        115                 120                 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro    130                 135                 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145                 150                 155                 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser                165                 170                 175Pro Ser Tyr Ala Ser             180

The corresponding cDNA sequence coding for the mature FGF21 polypeptide(SEQ ID NO:3) is shown below (SEQ ID NO:4):

1 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 61ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 121gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 181ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 301gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 421ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 481ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 541tcctga

The fusion proteins of the invention may comprise protein variants ormutants of the wild-type proteins listed herein, e.g., FGF21 variants.As used herein, the terms “protein variant,” “human variant,”“polypeptide or protein variant,” “variant,” “mutant,” as well as anylike terms or specific versions thereof (e.g., “FGF21 protein variant,”“variant,” “FGF21 mutant,” etc.) define protein or polypeptide sequencesthat comprise modifications, truncations, other variants of naturallyoccurring (i.e., wild-type) protein or polypeptide counterparts orcorresponding native sequences. “Variant FGF21” or “FGF21 mutant,” forinstance, is described relative to the wild-type (i.e., naturallyoccurring) FGF21 protein as described herein.

Representative fusion protein sequences of the invention are listed inTable 1. The descriptions of said fusions include the FGF21 variant and,where applicable, a linker. The changes or substitutions employed by theFGF21 variant are numbered and described relative to wild-type FGF21. Byway of example, “Variant 101 (V101)” (SEQ ID NO:10) is an Fc-FGF21fusion with a two amino acid linker and the following substitutions maderelative to wild type FGF21: Q55C, A109T, G148C, K150R, P158S, P174L,S195A, P199G, G202A.

TABLE 1 FGF21 Variant Fc fusion proteins SEQ ID NO: Sequence Name* 7DKTHTCPPCP APEAAGGPSV FLFPPKPKDT Full Length N-termLMISRTPEVT CVVVDVSHED PEVKFNWYVD Fc-Fusion with 2 AA LinkerGVEVHNAKTK PREEQYNSTY RVVSVLTVLH (GS) and WI FGF21QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ VRQRYLYTDD AQQTEAHLEI REDGTVGGAADQSPESLLQL KALKPGVIQI LGVKTSRFLC QRPDGALYGS LHFDPEACSF RELLLEDGYNVYQSEAHGLP LHLPGNKSPH RDPAPRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDPLSMVGPSQGR SPSYAS 8 DKTHTCPPCP APEAAGGPSV FLFPPKPKDTFull Length N-term Fc- LMISRTPEVT CVVVDVSHED PEVKFNWYVDFusion with 15 AA GVEVHNAKTK PREEQYNSTY RVVSVLTVLH Linker (GGGGS x 3)QDWLNGKEYK CKVSNKALPA PIEKTISKAK between Fc and WT FGF21GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGGG GSGGGGSGGGGSDSSPLLQF GGQVRQRYLY TDDAQQTEAH LEIREDGTVG GAADQSPESL LQLKALKPGVIQILGVKTSR FLCQRPDGAL YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPGNKSPHRDPAPRG RARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS QGRSPSYAS 9DSSPLLQFGG QVRQRYLYTD DAQETEAHLE Variant #76 =ProteinIREDGTVGGA AHQSPESLLE LKALKPGVIQ with 9 total mutationsILGVKTSRFL CQKPDGALYG SLHFDPEACS relative to wild-typeFRELLLEDGY NVYQSEAHGL PLHLPGNRSP FGF21 (as inHCDPAPQGPA RFLPLPGLPP ALPEPPGILA WO01/018172)PQPPDVGSSD PLAMVGPSQG RSPSYAS 10 DKTHTCPPCP APEAAGGPSV FLFPPKPKDTVariant # 101 = N-term LMISRTPEVT CVVVDVSHED PEVKFNWYVDFc Fusion with the 2 AA GVEVHNAKTK PREEQYNSTY RVVSVLTVLHlinker (GS) between Fc QDWLNGKEYK CKVSNKALPA PIEKTISKAK and FGF21 =(Q55C, GQPREPQVYT LPPSREEMTK NQVSLTCLVK A109T, G148C, K150R,GFYPSDIAVE WESNGQPENN YKTTPPVLDS P158S, 5195A, P199G,DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE G202A) ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQVRQRYLYTDD ACQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLCQRPDGTLYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPASRGPARFLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS 11DKTHTCPPCP APEAAGGPSV FLFPPKPKDT Variant # 103 = N-termLMISRTPEVT CVVVDVSHED PEVKFNWYVD Fc Fusion with the 2 AAGVEVHNAKTK PREEQYNSTY RVVSVLTVLH linker (GS) = (Q55C,QDWLNGKEYK CKVSNKALPA PIEKTISKAK R105K, G148C, K150R,GQPREPQVYT LPPSREEMTK NQVSLTCLVK P158S, S195A, P199G,GFYPSDIAVE WESNGQPENN YKTTPPVLDS G202A) DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ VRQRYLYTDD ACQTEAHLEI REDGTVGGAADQSPESLLQL KALKPGVIQI LGVKTSRFLC QKPDGALYGS LHFDPEACSF RELLLEDGYNVYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDPLAMVGGSQAR SPSYAS 12 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT Variant #188 =V103 LMISRTPEVT CVVVDVSHED PEVKFNWYVD with 15 AA LinkerGVEVHNAKTK PREEQYNSTY RVVSVLTVLH (GGGGS × 3) between FcQDWLNGKEYK CKVSNKALPA PIEKTISKAK and FGF21 =GQPREPQVYT LPPSREEMTK NQVSLTCLVK (Q55C, R105K, G148C,GFYPSDIAVE WESNGQPENN YKTTPPVLDS K150R, P158S, S195A,DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE P199G, G202A)ALHNHYTQKS LSLSPGKGGG GSGGGGSGGG GSDSSPLLQF GGQVRQRYLY TDDACQTEAHLEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQKPDGAL YGSLHFDPEACSFRELLLED GYNVYQSEAH GLPLHLPCNR SPHRDPASRG PARFLPLPGL PPALPEPPGILAPQPPDVGS SDPLAMVGGS QARSPSYAS 13 DKTHTCPPCP APEAAGGPSV FLFPPKPKDTVariant #204 = V101 LMISRTPEVT CVVVDVSHED PEVKFNWYVD with 15 AA LinkerGVEVHNAKTK PREEQYNSTY RVVSVLTVLH (GGGGS × 3) between FcQDWLNGKEYK CKVSNKALPA PIEKTISKAK and FGF21 = (Q55C,GQPREPQVYT LPPSREEMTK NQVSLTCLVK A109T, G148C, K150R,GFYPSDIAVE WESNGQPENN YKTTPPVLDS P158S, S195A, P199G,DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE G202A) ALHNHYTQKS LSLSPGKGGG GSGGGGSGGGGSDSSPLLQF GGQVRQRYLY TDDACQTEAH LEIREDGTVG GAADQSPESL LQLKALKPGVIQILGVKTSR FLCQRPDGTL YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPCNRSPHRDPASRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLAMVGGS QARSPSYAS *Notethat the FGF21 wild-type sequence in this table refers to NCBI referencesequence number NP_061986.1 (SEQ ID NO: 1) unless otherwise specified.All mutations in the FGF21 moiety and corresponding amino acid numberingof said mutations refers back to (SEQ ID NO: 1) not to the full-lengthsequences in this table which may also include Fc and linker regions.

The variants or mutants used in the fusion proteins of the invention,e.g., variants of wild-type FGF21, feature at least one substituted,added, and/or removed amino acid relative to the wild-type protein.Additionally, the variants may include N- and/or C-terminal truncationsrelative to the wild-type proteins. Generally speaking, a variantpossesses some modified property, structural or functional, of thewild-type protein. For example, the variant may have enhanced orimproved physical stability in concentrated solutions (e.g., lesshydrophobic mediated aggregation), enhanced or improved plasma stabilitywhen incubated with blood plasma or enhanced or improved bioactivitywhile maintaining a favorable bioactivity profile.

Acceptable amino acid substitutions and modifications which constitutedifferences between the portions of the fusion proteins of the inventionand their wild-type comparator proteins include, but are not limited to,one or more amino acid substitutions, including substitutions withnon-naturally occurring amino acid analogs, and truncations. Thus, thefusion proteins of the invention (e.g., the fusion proteins of theinvention) include, but are not limited to, site-directed mutants,truncated polypeptides, proteolysis-resistant mutants,aggregation-reducing mutants, combination mutants, and fusion proteins,as described herein.

One skilled in the art of expression of proteins will recognize thatmethionine or methionine-arginine sequence can be introduced at theN-terminus of any of the fusion proteins of the invention, forexpression in E. coli, and are contemplated within the context of thisinvention.

The fusion proteins of the invention may possess increased compatibilitywith pharmaceutical preservatives (e.g., m-cresol, phenol, benzylalcohol), thus enabling the preparation of a preserved pharmaceuticalformulation that maintains the physiochemical properties and biologicalactivity of the protein during storage. Accordingly, variants withenhanced pharmaceutical stability relative to wild-type, have improvedphysical stability in concentrated solutions under both physiologicaland preserved pharmaceutical formulation conditions, while maintainingbiological potency. By way of non-limiting example, the fusion proteinsof the invention may be more resistant to proteolysis and enzymaticdegradation; may have improved stability; and may be less likely toaggregate, than their wild-type counterparts or corresponding nativesequence. As used herein, these terms are not mutually exclusive orlimiting, it being entirely possible that a given variant has one ormore modified properties of the wild-type protein.

The invention also encompasses nucleic acid molecules encoding thefusion proteins of the invention, comprising, for example, an FGF21amino acid sequence that is at least about 95% identical to the aminoacid sequence of SEQ ID NO:3, but wherein specific residues conferring adesirable property to the FGF21 protein variant, e.g., improved potencyto FGF21-receptors, proteolysis-resistance, increased half life oraggregation-reducing properties and combinations thereof have not beenfurther modified. In other words, with the exception of residues in theFGF21 mutant sequence that have been modified in order to conferproteolysis-resistance, aggregation-reducing, or other properties, about5% (alternately 4%, alternately 3%, alternately 2%, alternately 1%) ofall other amino acid residues in the FGF21 mutant sequence can bemodified. Such FGF21 mutants possess at least one activity of thewild-type FGF21 polypeptide.

The invention also encompasses a nucleic acid molecule comprising anucleotide sequence that is at least about 85%, identical, and morepreferably, at least about 90 to 95% identical to the nucleotidesequence of SEQ ID NO:2 or SEQ ID NO:4, but wherein the nucleotidesencoding amino acid residues conferring the encoded protein'sproteolysis-resistance, aggregation-reducing or other properties havenot been further modified. In other words, with the exception ofnucleotides that encode residues in the FGF21 mutant sequences that havebeen modified in order to confer proteolysis-resistance,aggregation-reducing, or other properties, about 15%, and morepreferably about 10 to 5% of all other nucleotides in the mutantsequence can be modified. Such nucleic acid molecules encode proteinspossessing at least one activity of their wild-type counterparts.

Provided herein are methods used to generate the fusion proteins of theinvention, wherein such methods involve site-specific modification andnon-site-specific modification of the wild-type versions of the proteins(e.g., the FGF21 wild-type protein as described herein), e.g.,truncations of the wild-type proteins, and the site-specificincorporation of amino acids at positions of interest within thewild-type proteins. Said modifications enhance the biological propertiesof the fusion proteins of the invention relative to the wild-typeproteins, as well as, in some cases, serving as points of attachmentfor, e.g., labels and protein half-life extension agents, and forpurposes of affixing said variants to the surface of a solid support.Related embodiments of the invention are methods of producing cellscapable of producing said Fusion Proteins of the invention, and ofproducing vectors containing DNA encoding said variants.

In certain embodiments, such modifications, e.g., site-specificmodifications, are used to attach conjugates, e.g., PEG groups toproteins, polypeptides, and/or peptides of the invention, for purposesof, e.g., extending half-life or otherwise improving the biologicalproperties of said proteins, polypeptides, and/or peptides. Saidtechniques are described further herein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used to attach other polymers, small molecules andrecombinant protein sequences that extend half-life of the protein ofthe invention. One such embodiment includes the attachment of fattyacids or specific albumin binding compounds to proteins, polypeptides,and/or peptides. In other embodiments, the modifications are made at aparticular amino acid type and may be attached at one or more sites onthe protein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used as means of attachment for the production ofwild-type and/or variant multimers, e.g., dimers (homodimers orheterodimers) or trimers or tetramers. These multimeric proteinmolecules may additionally have groups such as PEG, sugars, and/orPEG-cholesterol conjugates attached or be fused either amino-terminallyor carboxy-terminally to other proteins such as Fc, Human Serum Albumin(HSA), etc.

In other embodiments, such site-specific modifications are used toproduce proteins, polypeptides and/or peptides wherein the position ofthe site-specifically incorporated pyrrolysine or pyrrolysine analogueor non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe)allows for controlled orientation and attachment of such proteins,polypeptides and/or peptides onto a surface of a solid support or tohave groups such as PEG, sugars and/or PEG-cholesterol conjugatesattached.

In other embodiments, such site-specific modifications are used tosite-specifically cross-link proteins, polypeptides and/or peptidesthereby forming hetero-oligomers including, but not limited to,heterodimers and heterotrimers. In other embodiments, such site-specificmodifications are used to site-specifically cross-link proteins,polypeptides and/or peptides thereby forming protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates. In other embodiments, a site specificmodification may include a branching point to allow more than one typeof molecule to be attached at a single site of a protein, polypeptide orpeptide.

In other embodiments, the modifications listed herein can be done in anon-site-specific manner and result in protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates of the invention.

Definitions

Various definitions are used throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art.

As used herein, the term “FGF21” refers to a member of the fibroblastgrowth factor (FGF) protein family. An amino acid sequence of FGF21(GenBank Accession No. NP_061986.1) is set forth as SEQ ID NO:1, thecorresponding polynucleotide sequence of which is set forth as SEQ IDNO:2 (NCBI reference sequence number NM_019113.2). “FGF21 variant,”“FGF21 mutant,” and similar terms describe modified version of the FGF21protein, e.g., with constituent amino acid residues deleted, added,modified, or substituted.

As used herein, the term “FGF21 receptor” refers to a receptor for FGF21(Kharitonenkov, A, et al. (2008) Journal of Cellular Physiology 215:1-7;Kurosu, H et al. (2007) JBC 282:26687-26695; Ogawa, Y et al. (2007) PNAS104:7432-7437).

The term “FGF21 polypeptide” refers to a naturally-occurring polypeptideexpressed in humans. For purposes of this disclosure, the term “FGF21polypeptide” can be used interchangeably to refer to any full-lengthFGF21 polypeptide, e.g., SEQ ID NO:1, which consists of 209 amino acidresidues and which is encoded by the nucleotide sequence of SEQ ID NO:2;any mature form of the polypeptide, which consists of 181 amino acidresidues, and in which the 28 amino acid residues at the amino-terminalend of the full-length FGF21 polypeptide (i.e., which constitute thesignal peptide) have been removed.

“Variant 76,” as used herein, is an FGF21 protein variant, featuring a40 kDa branched PEG linked through Cys154, and eight point mutationsrelative to the 177 amino acid wild-type protein. Synthesis of thevariant is described in greater detail herein, and the protein sequenceis represented in Table 1 and SEQ ID NO:9.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the present invention that (1) has been separated from atleast about 50 percent of proteins, lipids, carbohydrates, or othermaterials with which it is naturally found when total nucleic acid isisolated from the source cells, (2) is not linked to all or a portion ofa polynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecules or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “operably linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Elementsof fusions proteins may be operably linked to one another so as to allowthe fusion protein to function as if it were a naturally occurring,endogenous protein, and/or to combine disparate elements of said fusionproteins in a synergistic fashion.

On a nucleotide level, a flanking sequence operably linked to a codingsequence may be capable of effecting the replication, transcriptionand/or translation of the coding sequence. For example, a codingsequence is operably linked to a promoter when the promoter is capableof directing transcription of that coding sequence. A flanking sequenceneed not be contiguous with the coding sequence, so long as it functionscorrectly. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” when used in connection with biologicalmaterials such as nucleic acid molecules, polypeptides, host cells, andthe like, refers to materials which are found in nature and are notmanipulated by man. Similarly, “non-naturally occurring” as used hereinrefers to a material that is not found in nature or that has beenstructurally modified or synthesized by man. When used in connectionwith nucleotides, the term “naturally occurring” refers to the basesadenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).When used in connection with amino acids, the term “naturally occurring”refers to the 20 conventional amino acids (i.e., alanine (A), cysteine(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine(G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine(M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine(S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), aswell as selenocysteine, pyrrolysine (Pyl, or O), andpyrroline-carboxy-lysine (Pcl, or Z).

Pyrrolysine (Pyl) is an amino acid naturally found within methylaminemethyltransferases of methanogenic archaea of the family Methanosarcina.Pyrrolysine is a lysine analogue co-translationally incorporated atin-frame UAG codons in the respective mRNA, and it is considered the22nd natural amino acid.

As described at least in PCT patent publication WO2010/48582 (applicantIRM, LLC), attempts to biosynthesize pyrrolysine (Pyl) in E. coliresulted in the formation of a “demethylated pyrrolysine,” referred toherein as pyrroline-carboxy-lysine, or Pcl. “Pcl,” as used herein,refers to either Pcl-A or Pcl-B.

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) FGF21 protein sequence or thesequences of the present invention. These include, but are not limitedto, modified amino acids and/or amino acid analogues that are not one ofthe 20 naturally occurring amino acids, selenocysteine, pyrrolysine(Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCTpatent publication WO2010/48582). Such non-natural amino acid residuescan be introduced by substitution of naturally occurring amino acids,and/or by insertion of non-natural amino acids into the naturallyoccurring (wild-type) FGF21 protein sequence or the sequences of theinvention. The non-natural amino acid residue also can be incorporatedsuch that a desired functionality is imparted to the FGF21 molecule, forexample, the ability to link a functional moiety (e.g., PEG). When usedin connection with amino acids, the symbol “U” shall mean “non-naturalamino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” requirea modified tRNA and a modified tRNA synthetase (RS) for incorporationinto a protein. These “selected” orthogonal tRNA/RS pairs are generatedby a selection process as developed by Schultz et al. or by random ortargeted mutation. As way of example, pyrroline-carboxy-lysine is a“natural amino acid” as it is generated biosynthetically by genestransferred from one organism into the host cells and as it isincorporated into proteins by using natural tRNA and tRNA synthetasegenes, while p-aminophenylalanine (See, Generation of a bacterium with a21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L,Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003 January29; 125(4):935-9) is an “unnatural amino acid” because, althoughgenerated biosynthetically, it is incorporated into proteins by a“selected” orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to,hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term “amino acid” also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, by way of example only, an α-carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group. Amino acid analoguesinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or their C-terminal carboxy group,their N-terminal amino group and/or their side-chain functional groupsare chemically modified. Such analogues include, but are not limited to,methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteinesulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alaninecarboxamide, homoserine, norleucine, and methionine methyl sulfonium.

The term “amino acid mimetics,” as used herein, refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but functions in a manner similarto a naturally occurring amino acid.

The term “biologically active variant” refers to any polypeptide variantused in the fusion proteins of the invention, e.g., as a constituentprotein of the fusions, that possesses an activity of its wild-type(e.g., naturally-occurring) protein or polypeptide counterpart, such asthe ability to modulate blood glucose, HbA1c, insulin, triglyceride, orcholesterol levels; increase pancreatic function; reduce lipid levels inliver; reduce body weight; and to improve glucose tolerance, energyexpenditure, or insulin sensitivity, regardless of the type or number ofmodifications that have been introduced into the polypeptide variant.Polypeptide variants possessing a somewhat decreased level of activityrelative to their wild-type versions can nonetheless be considered to bebiologically active polypeptide variants. A non-limiting representativeexample of a biologically active polypeptide variant of the invention isan FGF21 variant, which is modified after, and possesses similar orenhanced biological properties relative to, wild-type FGF21.

The terms “effective amount” and “therapeutically effective amount” eachrefer to the amount of a fusion protein of the invention used to supportan observable level of one or more biological activities of thewild-type polypeptide or protein counterparts, such as the ability tolower blood glucose, insulin, triglyceride or cholesterol levels; reduceliver triglyceride or lipid levels; reduce body weight; or improveglucose tolerance, energy expenditure, or insulin sensitivity. Forexample, a “therapeutically-effective amount” administered to a patientexhibiting, suffering, or prone to suffer from FGF21-associateddisorders (such as type 1 or type 2 diabetes mellitus, obesity, ormetabolic syndrome), is such an amount which induces, ameliorates orotherwise causes an improvement in the pathological symptoms, diseaseprogression, physiological conditions associated with or resistance tosuccumbing to the aforementioned disorders. For the purposes of thepresent invention a “subject” or “patient” is preferably a human, butcan also be an animal, more specifically, a companion animal (e.g.,dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs,horses, and the like) and laboratory animals (e.g., rats, mice, guineapigs, and the like).

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of afusion protein of the invention.

The term “antigen” refers to a molecule or a portion of a molecule thatis capable of being bound by an antibody, and additionally that iscapable of being used in an animal to produce antibodies that arecapable of binding to an epitope of that antigen. An antigen may haveone or more epitopes.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody or produced by other means, whether in monomeric ormultimeric form, and can contain the hinge region. The originalimmunoglobulin source of the native Fc is preferably of human origin andcan be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc molecules are made up of monomeric polypeptides that can belinked into dimeric or multimeric forms by covalent (i.e., disulfidebonds) and non-covalent association. The number of intermoleculardisulfide bonds between monomeric subunits of native Fc molecules rangesfrom 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass(e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc isa disulfide-bonded dimer resulting from papain digestion of an IgG (seeEllison et al., 1982, Nucleic Acids Res. 10: 4071-9). The term “nativeFc” as used herein is generic to the monomeric, dimeric, and multimericforms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn (neonatal Fc receptor). International Publication Nos. WO97/34631 and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. Thus, the term “Fc variant” can comprise a molecule orsequence that is humanized from a non-human native Fc. Furthermore, anative Fc comprises regions that can be removed because they providestructural features or biological activity that are not required for thefusion molecules of the fusion proteins of the invention. Thus, the term“Fc variant” comprises a molecule or sequence that lacks one or morenative Fc sites or residues, or in which one or more Fc sites orresidues has be modified, that affect or are involved in: (1) disulfidebond formation, (2) incompatibility with a selected host cell, (3)N-terminal heterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variants and sequencesas defined above. As with Fc variants and native Fc molecules, the term“Fc domain” includes molecules in monomeric or multimeric form, whetherdigested from whole antibody or produced by other means. In someembodiments of the present invention, an Fc domain can be fused to FGF21or a FGF21 mutant (including a truncated form of FGF21 or a FGF21mutant) via, for example, a covalent bond between the Fc domain and theFGF21 sequence. Such fusion proteins can form multimers via theassociation of the Fc domains and both these fusion proteins and theirmultimers are an aspect of the present invention.

The term “modified Fc fragment”, as used herein, shall mean an Fcfragment of an antibody comprising a modified sequence. The Fc fragmentis a portion of an antibody comprising the CH2, CH3 and part of thehinge region. The modified Fc fragment can be derived from, for example,IgGI, IgG2, IgG3, or IgG4. FcLALA is a modified Fc fragment with a LALAmutation (L234A, L235A), which triggers ADCC with lowered efficiency,and binds and activates human complement weakly. Hessell et al. 2007Nature 449:101-104. Additional modifications to the Fc fragment aredescribed in, for example, U.S. Pat. No. 7,217,798.

The term “heterologous” means that these domains are not naturally foundassociated with constant regions of an antibody. In particular, suchheterologous binding domains do not have the typical structure of anantibody variable domain consisting of 4 framework regions, FR1, FR2,FR3 and FR4 and the 3 complementarity determining regions (CDRs)in-between. Each arm of the fusobody therefore comprises a first singlechain polypeptides comprising a first binding domain covalently linkedat the N-terminal part of a constant C_(H)1 heavy chain region of anantibody, and a second single chain polypeptide comprising a secondbinding domain covalently linked at the N-terminal part of a constantC_(L) light chain of an antibody. The covalent linkage may be direct,for example via peptidic bound or indirect, via a linker, for example apeptidic linker. The two heterodimers of the fusobody are covalentlylinked, for example, by at least one disulfide bridge at their hingeregion, like an antibody structure. Examples of molecules with afusobody structure have been described in the art, in particular,fusobodies comprising ligand binding region of heterodimeric receptor(see for example international patent publications WO01/46261 andWO11/076781).

The term “polyethylene glycol” or “PEG” refers to a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties.

The term “FGF21-associated disorders,” and terms similarly used herein,includes obesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, acute myocardialinfarction, hypertension, cardiovascular disease, atherosclerosis,peripheral arterial disease, stroke, heart failure, coronary heartdisease, kidney disease, diabetic complications, neuropathy,gastroparesis, disorders associated with severe inactivating mutationsin the insulin receptor, and other metabolic disorders.

The term “disorders associated with severe inactivating mutations in theinsulin receptor,” and terms similarly used herein, describe conditionsin subjects afflicted with mutations in the insulin receptor (orpossible proteins directly downstream from it) which cause severeinsulin resistance but are often (though not always) seen without theobesity common in Type 2 diabetes mellitus. In many ways, subjectsafflicted with these conditions manifest hybrid symptoms of Type 1diabetes mellitus and Type 2 diabetes mellitus. Subjects therebyafflicted fall into several categories of roughly increasing severity,including: Type A Insulin Resistance, Type C Insulin Resistance (AKAHAIR-AN Syndrome), Rabson-Mendenhall Syndrome and finally Donohue'sSyndrome or Leprechaunism. These disorders are associated with very highendogenous insulin levels, and very often, hyperglycemia. Subjectsthereby afflicted also present with various clinical features associatedwith “insulin toxicity,” including hyperandrogenism, polycystic ovariansyndrome (PCOS), hirsuitism, and acanthosis nigricans (excessive growthand pigmentation) in the folds of the skin.

“Type 2 diabetes mellitus” is a condition characterized by excessglucose production in spite of the availability of insulin, andcirculating glucose levels remain excessively high as a result ofinadequate glucose clearance.

“Type 1 diabetes mellitus” is a condition characterized by high bloodglucose levels caused by total lack of insulin. This occurs when thebody's immune system attacks the insulin-producing beta cells in thepancreas and destroys them. The pancreas then produces little or noinsulin.

“Glucose intolerance” or Impaired Glucose Tolerance (IGT) is apre-diabetic state of dysglycemia that is associated with increased riskof cardiovascular pathology. The pre-diabetic condition prevents asubject from moving glucose into cells efficiently and utilizing it asan efficient fuel source, leading to elevated glucose levels in bloodand some degree of insulin resistance.

“Hyperglycemia” is defined as an excess of sugar (glucose) in the blood.

“Hypoglycemia”, also called low blood sugar, occurs when your bloodglucose level drops too low to provide enough energy for your body'sactivities.

“Hyperinsulinemia” is defined as a higher-than-normal level of insulinin the blood.

“Insulin resistance” is defined as a state in which a normal amount ofinsulin produces a subnormal biologic response.

“Obesity,” in terms of the human subject, can be defined as that bodyweight over 20 percent above the ideal body weight for a givenpopulation (R. H. Williams, Textbook of Endocrinology, 1974, p.904-916).

“Diabetic complications” are problems, caused by high blood glucoselevels, with other body functions such as kidneys, nerves(neuropathies), feet (foot ulcers and poor circulation) and eyes (e.g.retinopathies). Diabetes also increases the risk for heart disease andbone and joint disorders. Other long-term complications of diabetesinclude skin problems, digestive problems, sexual dysfunction andproblems with teeth and gums.

“Metabolic syndrome” can be defined as a cluster of at least three ofthe following signs: abdominal fat—in most men, a 40-inch waist orgreater; high blood sugar—at least 110 milligrams per deciliter (mg/dl)after fasting; high triglycerides—at least 150 mg/dL in the bloodstream;low HDL—less than 40 mg/dl; and, blood pressure of 130/85 mmHg orhigher.

“Pancreatitis” is inflammation of the pancreas.

“Dyslipidemia” is a disorder of lipoprotein metabolism, includinglipoprotein overproduction or deficiency. Dyslipidemias may bemanifested by elevation of the total cholesterol, low-densitylipoprotein (LDL) cholesterol and triglyceride concentrations, and adecrease in high-density lipoprotein (HDL) cholesterol concentration inthe blood.

“Nonalcoholic fatty liver disease (NAFLD)” is a liver disease, notassociated with alcohol consumption, characterized by fatty change ofhepatocytes.

“Nonalcoholic steatohepatitis (NASH)” is a liver disease, not associatedwith alcohol consumption, characterized by fatty change of hepatocytes,accompanied by intralobular inflammation and fibrosis.

“Hypertension” or high blood pressure that is a transitory or sustainedelevation of systemic arterial blood pressure to a level likely toinduce cardiovascular damage or other adverse consequences. Hypertensionhas been arbitrarily defined as a systolic blood pressure above 140 mmHgor a diastolic blood pressure above 90 mmHg.

“Cardiovascular diseases” are diseases related to the heart or bloodvessels.

“Acute myocardial infarction” occurs when there is interruption of theblood supply to a part of the heart. The resulting ischemia and oxygenshortage, if left untreated for a sufficient period of time, can causedamage or death (infarction) of the heart muscle tissue (myocardium).

“Peripheral arterial disease” occurs when plaque builds up in thearteries that carry blood to the head, organs and limbs. Over time,plaque can harden and narrow the arteries which limits the flow ofoxygen-rich blood to organs and other parts of the body.

“Atherosclerosis” is a vascular disease characterized by irregularlydistributed lipid deposits in the intima of large and medium-sizedarteries, causing narrowing of arterial lumens and proceeding eventuallyto fibrosis and calcification. Lesions are usually focal and progressslowly and intermittently. Limitation of blood flow accounts for mostclinical manifestations, which vary with the distribution and severityof lesions.

“Stroke” is any acute clinical event, related to impairment of cerebralcirculation, that lasts longer than 24 hours. A stroke involvesirreversible brain damage, the type and severity of symptoms dependingon the location and extent of brain tissue whose circulation has beencompromised.

“Heart failure”, also called congestive heart failure, is a condition inwhich the heart can no longer pump enough blood to the rest of the body.

“Coronary heart disease”, also called coronary artery disease, is anarrowing of the small blood vessels that supply blood and oxygen to theheart.

“Kidney disease” or nephropathy is any disease of the kidney. Diabeticnephropathy is a major cause of morbidity and mortality in people withtype 1 or type 2 diabetes mellitus.

“Neuropathies” are any diseases involving the cranial nerves or theperipheral or autonomic nervous system.

“Gastroparesis” is weakness of gastric peristalsis, which results indelayed emptying of the bowels.

The critically ill patients encompassed by the present inventiongenerally experience an unstable hypermetabolic state. This unstablemetabolic state is due to changes in substrate metabolism, which maylead to relative deficiencies in some nutrients. Generally there is anincreased oxidation of both fat and muscle.

Moreover, critically ill patients are preferably patients thatexperience systemic inflammatory response syndrome or respiratorydistress. A reduction in morbidity means reducing the likelihood that acritically ill patient will develop additional illnesses, conditions, orsymptoms or reducing the severity of additional illnesses, conditions,or symptoms. For example reducing morbidity may correspond to a decreasein the incidence of bacteremia or sepsis or complications associatedwith multiple organ failure.

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise. Thus, forexample, reference to “an antibody” includes a mixture of two or moresuch antibodies.

As used herein, the term “about” refers to +/−20%, more preferably,+/−10%, or still more preferably, +/−5% of a value.

The terms “polypeptide” and “protein”, are used interchangeably andrefer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, naturally and non-naturallyoccurring amino acids, chemically or biochemically modified orderivatized amino acids, and polypeptides having modified peptidebackbones. The term includes fusion proteins, including, but not limitedto, fusion proteins with a heterologous amino acid sequence, fusionswith heterologous and homologous leader sequences, with or withoutN-terminal methionine residues; immunologically tagged proteins; and thelike.

The terms “individual”, “subject”, “host” and “patient” are usedinterchangeably and refer to any subject for whom diagnosis, treatment,or therapy is desired, particularly humans. Other subjects may includecattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and thelike. In some preferred embodiments the subject is a human.

As used herein, the term “sample” refers to biological material from apatient. The sample assayed by the present invention is not limited toany particular type. Samples include, as non-limiting examples, singlecells, multiple cells, tissues, tumors, biological fluids, biologicalmolecules, or supernatants or extracts of any of the foregoing. Examplesinclude tissue removed for biopsy, tissue removed during resection,blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous,and stool samples. The sample used will vary based on the assay format,the detection method and the nature of the tumors, tissues, cells orextracts to be assayed. Methods for preparing samples are well known inthe art and can be readily adapted in order to obtain a sample that iscompatible with the method utilized.

As used herein, the term “biological molecule” includes, but is notlimited to, polypeptides, nucleic acids, and saccharides.

As used herein, the term “modulating” refers to a change in the qualityor quantity of a gene, protein, or any molecule that is inside, outside,or on the surface of a cell. The change can be an increase or decreasein expression or level of the molecule. The term “modulates” alsoincludes changing the quality or quantity of a biologicalfunction/activity including, without limitation, the ability to lowerblood glucose, insulin, triglyceride, or cholesterol levels; to reduceliver lipid or liver triglyceride levels; to reduce body weight; and toimprove glucose tolerance, energy expenditure, or insulin sensitivity.

As used herein, the term “modulator” refers to a composition thatmodulates one or more physiological or biochemical events associatedwith an FGF21-associated disorder, such as type 1 or type 2 diabetesmellitus or a metabolic condition like obesity. Said events include butare not limited to the ability to lower blood glucose, insulin,triglyceride, or cholesterol levels; to reduce liver lipid or livertriglyceride levels; to reduce body weight; and to improve glucosetolerance, energy expenditure, or insulin sensitivity.

A “gene product” is a biopolymeric product that is expressed or producedby a gene. A gene product may be, for example, an unspliced RNA, anmRNA, a splice variant mRNA, a polypeptide, a post-translationallymodified polypeptide, a splice variant polypeptide etc. Also encompassedby this term are biopolymeric products that are made using an RNA geneproduct as a template (i.e. cDNA of the RNA). A gene product may be madeenzymatically, recombinantly, chemically, or within a cell to which thegene is native. In some embodiments, if the gene product isproteinaceous, it exhibits a biological activity. In some embodiments,if the gene product is a nucleic acid, it can be translated into aproteinaceous gene product that exhibits a biological activity.

“Modulation of FGF21 activity,” as used herein, refers to an increase ordecrease in FGF21 activity that can be a result of, for example,interaction of an agent with an FGF21 polynucleotide or polypeptide,inhibition of FGF21 transcription and/or translation (e.g., throughantisense or siRNA interaction with the FGF21 gene or FGF21 transcript,through modulation of transcription factors that facilitate FGF21expression), and the like. For example, modulation of a biologicalactivity refers to an increase or a decrease in a biological activity.FGF21 activity can be assessed by means including, without limitation,assaying blood glucose, insulin, triglyceride, or cholesterol levels ina subject, assessing FGF21 polypeptide levels, or by assessing FGF21transcription levels. Comparisons of FGF21 activity can also beaccomplished by, e.g., measuring levels of an FGF21 downstreambiomarker, and measuring increases in FGF21 signaling. FGF21 activitycan also be assessed by measuring: cell signaling; kinase activity;glucose uptake into adipocytes; blood insulin, triglyceride, orcholesterol level fluctuations; liver lipid or liver triglyceride levelchanges; interactions between FGF21 and an FGF21 receptor; orphosphorylation of an FGF21 receptor. In some embodimentsphosphorylation of an FGF21 receptor can be tyrosine phosphorylation. Insome embodiments modulation of FGF21 activity can cause modulation of anFGF21-related phenotype.

Comparisons of FGF21 activity can also be accomplished by, e.g.,measuring levels of an FGF21 downstream biomarker, and measuringincreases in FGF21 signaling. FGF21 activity can also be assessed bymeasuring: cell signaling; kinase activity; glucose uptake intoadipocytes; blood insulin, triglyceride, or cholesterol levelfluctuations; liver lipid or liver triglyceride level changes;interactions between FGF21 and a receptor (FGFR-1c, FGFR-2c, orFGFR-3c); or phosphorylation of an FGF21 receptor. In some embodimentsphosphorylation of an FGF21 receptor can be tyrosine phosphorylation. Insome embodiments modulation of FGF21 activity can cause modulation of anFGF21-related phenotype.

A “FGF21 downstream biomarker,” as used herein, is a gene or geneproduct, or measurable indicia of a gene or gene product. In someembodiments, a gene or activity that is a downstream marker of FGF21exhibits an altered level of expression, or in a vascular tissue. Insome embodiments, an activity of the downstream marker is altered in thepresence of an FGF21 modulator. In some embodiments, the downstreammarkers exhibit altered levels of expression when FGF21 is perturbedwith an FGF21 modulator of the present invention. FGF21 downstreammarkers include, without limitation, glucose or 2-deoxy-glucose uptake,pERK and other phosphorylated or acetylated proteins or NAD levels.

As used herein, the term “up-regulates” refers to an increase,activation or stimulation of an activity or quantity. For example, inthe context of the present invention, FGF21 modulators may increase theactivity of an FGF21 receptor. In one embodiment, one or more FGFR-1c,FGFR-2c, or FGFR-3c may be upregulated in response to an FGF21modulator. Upregulation can also refer to an FGF21-related activity,such as e.g., the ability to lower blood glucose, insulin, triglyceride,or cholesterol levels; to reduce liver lipid or triglyceride levels; toreduce body weight; to improve glucose tolerance, energy expenditure, orinsulin sensitivity; or to cause phosphorylation of an FGF21 receptor;or to increase an FGF21 downstream marker. The FGFR21 receptor can beone or more of FGFR-1c, FGFR-2c, or FGFR-3c. Up-regulation may be atleast 25%, at least 50%, at least 75%, at least 100%, at least 150%, atleast 200%, at least 250%, at least 400%, or at least 500% as comparedto a control.

As used herein, the term “N-terminus” refers to at least the first 20amino acids of a protein.

As used herein, the terms “N-terminal domain” and “N-terminal region”are used interchangeably and refer to a fragment of a protein thatbegins at the first amino acid of the protein and ends at any amino acidin the N-terminal half of the protein. For example, the N-terminaldomain of FGF21 is from amino acid 1 of SEQ ID NO:1 to any amino acidbetween about amino acids 10 and 105 of SEQ ID NO:1.

As used herein, the term “C-terminus” refers to at least the last 20amino acids of a protein.

As used herein, the terms “C-terminal domain” and “C-terminal region”are used interchangeably and refer to a fragment of a protein thatbegins at any amino acid in the C-terminal half of the protein and endsat the last amino acid of the protein. For example, the C-terminaldomain of FGF21 begins at any amino acid from amino acid 105 to aboutamino acid 200 of SEQ ID NO:1 and ends at amino acid 209 of SEQ ID NO:1.

The term “domain” as used herein refers to a structural part of abiomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof and may also incorporate a portion of a biomolecule that isdistinct from a particular region, in addition to all or part of thatregion.

As used herein, the term “signal domain” (also called “signal sequence”or “signal peptide”) refers to a peptide domain that resides in acontinuous stretch of amino acid sequence at the N-terminal region of aprecursor protein (often a membrane-bound or secreted protein) and isinvolved in post-translational protein transport. In many cases thesignal domain is removed from the full-length protein by specializedsignal peptidases after the sorting process has been completed. Eachsignal domain specifies a particular destination in the cell for theprecursor protein. The signal domain of FGF21 is amino acids 1-28 of SEQID NO:1.

As used herein, the term “receptor binding domain” refers to any portionor region of a protein that contacts a membrane-bound receptor protein,resulting in a cellular response, such as a signaling event.

As used herein, the term “ligand binding domain” refers to any portionor region of a fusion protein of the invention retaining at least onequalitative binding activity of a corresponding native sequence.

The term “region” refers to a physically contiguous portion of theprimary structure of a biomolecule. In the case of proteins, a region isdefined by a contiguous portion of the amino acid sequence of thatprotein. In some embodiments a “region” is associated with a function ofthe biomolecule.

The term “fragment” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a portion is defined by a contiguous portion of the amino acidsequence of that protein and refers to at least 3-5 amino acids, atleast 8-10 amino acids, at least 11-15 amino acids, at least 17-24 aminoacids, at least 25-30 amino acids, and at least 30-45 amino acids. Inthe case of oligonucleotides, a portion is defined by a contiguousportion of the nucleic acid sequence of that oligonucleotide and refersto at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, andat least 90-130 nucleotides. In some embodiments, portions ofbiomolecules have a biological activity. In the context of the presentinvention, FGF21 polypeptide fragments do not comprise the entire FGF21polypeptide sequence set forth in SEQ ID NO:1.

A “native sequence” polypeptide is one that has the same amino acidsequence as a polypeptide derived from nature. Such native sequencepolypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. Thus, a native sequence polypeptide canhave the amino acid sequence of naturally occurring human polypeptide,murine polypeptide, or polypeptide from any other mammalian species.

As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by a homology, at the nucleotide level or aminoacid level, of at least a specified percentage and is usedinterchangeably with “sequence identity.” Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. Homologous amino acid sequences includethose amino acid sequences which contain conservative amino acidsubstitutions and which polypeptides have the same binding and/oractivity. In some embodiments, a nucleotide or amino acid sequence ishomologous if it has at least 60% or greater, up to 99%, identity with acomparator sequence. In some embodiments, a nucleotide or amino acidsequence is homologous if it shares one or more, up to 60,nucleotide/amino acid substitutions, additions, or deletions with acomparator sequence. In some embodiments, the homologous amino acidsequences have no more than 5 or no more than 3 conservative amino acidsubstitutions.

Percent homology or identity can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for UNIX,Genetics Computer Group, University Research Park, Madison Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482-489). In some embodiments, homology betweenthe probe and target is between about 75% to about 85%. In someembodiments, nucleic acids have nucleotides that are at least about 95%,about 97%, about 98%, about 99% and about 100% homologous to SEQ IDNO:2, or a portion thereof.

Homology may also be at the polypeptide level. In some embodiments,constituent polypeptides of the fusion proteins of the invention may beat least 95% homologous to their full length wild-type counterparts orcorresponding native sequences, or to portions thereof. The degree orpercentage identity of Fusion Proteins of the invention, or portionsthereof, and different amino acid sequences is calculated as the numberof exact matches in an alignment of the two sequences divided by thelength of the “invention sequence” or the “foreign sequence”, whicheveris shortest. The result is expressed as percent identity.

As used herein, the term “mixing” refers to the process of combining oneor more compounds, cells, molecules, and the like together in the samearea. This may be performed, for example, in a test tube, petri dish, orany container that allows the one or more compounds, cells, ormolecules, to be mixed.

As used herein, the term “substantially purified” refers to a compound(e.g., either a polynucleotide or a polypeptide or an antibody) that isremoved from its natural environment and is at least 60% free, at least75% free, and at least 90% free from other components with which it isnaturally associated.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent, such as antibodies or apolypeptide, genes, and other therapeutic agents. The term refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers in therapeuticcompositions can include liquids such as water, saline, glycerol andethanol. Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, can also be present in suchvehicles.

Enhancing the Physical Stability of the Fusion Proteins of the Invention

Naturally occurring disulfide bonds, as provided by cysteine residues,generally increase thermodynamic stability of proteins. Successfulexamples of increased thermodynamic stability, as measured in increaseof the melting temperature, are multiple disulfide-bonded mutants of theenzymes T4 lysozyme (Matsumura et al., PNAS 86:6562-6566 (1989)) andbarnase (Johnson et al., J. Mol. Biol. 268:198-208 (1997)). An aspect ofthe present invention is an enhancement of the physical stability ofFGF21 in the presence of a preservative, achieved by the presence ofdisulfide bonds within the variants, which constrain the flexibility ofwild-type FGF21 and thereby limit access of the preservative to thehydrophobic core of the protein.

The second aspect of the present invention therefore provides variantsof human FGF21, or a biologically active peptide thereof, with enhancedpharmaceutical stability engendered by the incorporation of additionaldisulfide bonds, e.g., via incorporating or substituting cysteineresidues into the wild-type FGF21 protein or the polypeptide and proteinvariants of the invention. One skilled in the art will recognize thatthe native cysteines, cysteine 103 and cysteine 121, could be utilizedas loci to introduce a novel disulfide bond that may impart improvedproperties, in addition to the suggested embodiments described herein.

These include fusion proteins which incorporate wild-type FGF-21 withthe substitution of a cysteine for two or more of the following:glutamine 46, arginine 47, tyrosine 48, leucine 49, tyrosine 50,threonine 51, aspartate 52, aspartate 53, alanine 54, glutamine 55,glutamine 56, threonine 57, glutamate 58, alanine 59, histidine 60,leucine 61, glutamate 62, isoleucine 63, valine 69, glycine 70, glycine71, alanine 72, alanine 73, leucine 144, histidine 145, leucine 146,proline 147, glycine 148, asparagine 149, lysine 150, serine 151,proline 152, histidine 153, arginine 154, aspartate 155, proline 156,alanine 157, proline 158, arginine 159, glycine 160, proline 161,alanine 162, arginine 163. phenylalanine 164, wherein the numbering ofthe amino acids is based on the full length 209 amino acid hFGF21sequence SEQ ID NO:1

Furthermore, fusion proteins of the invention may incorporate variantsof wild-type human FGF21, or a biologically active peptide thereof,which are enhanced with engineered disulfide bonds, in addition to thenaturally occurring one at Cys103-Cys121, are as follows:Gln46Cys-Ala59Cys, Gln46Cys-His60Cys, Gln46Cys-Leu61Cys,Gln46Cys-Glu62Cys, Gln46Cys-Ile63Cys, Arg47Cys-Ala59Cys,Arg47Cys-His60Cys, Arg47Cys-Leu61Cys, Arg47Cys-Glu62Cys,Arg47Cys-Ile63Cys, Tyr48Cys-Ala59Cys, Tyr48Cys-His60Cys,Tyr48Cys-Leu61Cys, Tyr48Cys-Glu62Cys, Tyr48Cys-Ile63Cys,Leu49Cys-Ala59Cys, Leu49Cys-His60Cys, Leu49Cys-Leu61Cys,Leu49Cys-Glu62Cys, Leu49Cys-Ile63Cys, Tyr50Cys-Ala59Cys,Tyr50Cys-His60Cys, Tyr50Cys-Lue61Cys, Tyr50Cys-Glu62Cys,Tyr50Cys-Ile63Cys, Leu144Cys-Gly160Cys, Leu144Cys-Pro161Cys,Leu144Cys-Ala162Cys, Leu144Cys-Arg163Cys, Leu144Cys-Phe164Cys,His145Cys-Gly160Cys, His145Cys-Pro161Cys, His145Cys-Ala162Cys,His145Cys-Arg163Cys, His145Cys-Phe164Cys, Leu146Cys-Gly160Cys,Leu146Cys-Pro161Cys, Leu146Cys-Ala162Cys, Leu146Cys-Arg163Cys,Leu146Cys-Phe164Cys, Pro147Cys-Gly160Cys, Pro147Cys-Pro161Cys,Pro147Cys-Ala162Cys, Pro147Cys-Arg163Cys, Pro147Cys-Phe164Cys,Gly148Cys-Gly160Cys, Gly148Cys-Pro161Cys, Gly148Cys-Ala162Cys,Gly148Cys-Arg163Cys, Gly148Cys-Phe164Cys, Thr57Cys-Val69Cys,Thr57Cys-Gly70Cys, Thr57Cys-Gly71Cys, Thr57Cys-Ala72Cys,Thr57Cys-Ala73Cys, Glu58Cys-Val69Cys, Glu58Cys-Glu70Cys,Glu58Cys-G71Cys, Glu58Cys-Ala72Cys, Glu58Cys-Ala73Cys,Ala59Cys-Val69Cys, Ala59Cys-Gly70Cys, Ala59Cys-Gly71Cys,Ala59Cys-Ala72Cys, Ala59Cys-Ala73Cys, His60Cys-Val69Cys,His60Cys-Gly70Cys, His60Cys-Gly71Cys, His60Cys-Ala72Cys,His60Cys-Ala73Cys, Leu61Cys-Val69Cys, Leu61Cys-Gly70Cys,Leu61Cys-Gly71Cys, Leu61Cys-Ala72Cys, Leu61Cys-Ala73Cys,Arg47Cys-Gly148Cys, Tyr48Cys-Gly148Cys, Leu49Cys-Gly148Cys,Tyr50Cys-Gly148Cys, Thr51Cys-Gly148Cys, Asp52Cys-Gly148Cys,Asp53Cys-Gly148Cys, Ala54Cys-Gly148Cys, Gln55Cys-Gly148Cys,Gln56Cys-Gly148Cys, Thr57Cys-Gly148Cys, Glu58Cys-Gly148Cys,Arg47Cys-Asn149Cys, Tyr48Cys-Asn149Cys, Leu49Cys-Asn149Cys,Tyr50Cys-Asn149Cys, Thr51Cys-Asn149Cys, Asp52Cys-Asn149Cys,Asp53Cys-Asn149Cys, Ala54Cys-Asn149Cys, Gln55Cys-Asn149Cys,Gln56Cys-Asn149Cys, Thr57Cys-Asn149Cys, Glu58Cys-Asn149Cys,Arg47Cys-Lys150Cys, Tyr48Cys-Lys150Cys, Leu49Cys-Lys150Cys,Tyr50Cys-Lys150Cys, Thr51Cys-Lys150Cys, Asp52Cys-Lys150Cys,Asp53Cys-Lys150Cys, Ala54Cys-Lys150Cys, Gln55Cys-Lys150Cys,Gln56Cys-Lys150Cys, Thr57Cys-Lys150Cys, Glu58Cys-Lys150Cys,Arg47Cys-Ser151Cys, Tyr48Cys-Ser151Cys, Leu49Cys-Ser151Cys,Tyr50Cys-Ser151Cys, Thr51Cys-Ser151Cys, Asp52Cys-Ser151Cys,Asp53Cys-Ser151Cys, Ala54Cys-Ser151Cys, Gln55Cys-Ser151Cys,Gln56Cys-Ser151Cys, Thr57Cys-Ser151Cys, Glu58Cys-Ser151Cys,Arg47Cys-Pro152Cys, Tyr48Cys-Pro152Cys, Leu49Cys-Pro152Cys,Tyr50Cys-Pro152Cys, Thr51Cys-Pro152Cys, Asp52Cys-Pro152Cys,Asp53Cys-Pro152Cys, Ala54Cys-Pro152Cys, Gln55Cys-Pro152Cys,Gln56Cys-Pro152Cys, Thr57Cys-Pro152Cys, Glu58Cys-Pro152Cys,Arg47Cys-His153Cys, Tyr48Cys-His153Cys, Leu49Cys-His153Cys,Tyr50Cys-His153Cys, Thr51Cys-His153Cys, Asp52Cys-His153Cys,Asp53Cys-His153Cys, Ala54Cys-His153Cys, Gln55Cys-His153Cys,Gln56Cys-His153Cys, Thr57Cys-His153Cys, Glu58Cys-His153Cys,Arg47Cys-Arg154Cys, Tyr48Cys-Arg154Cys, Leu49Cys-Arg154Cys,Tyr50Cys-Arg154Cys, Thr51Cys-Arg154Cys, Asp52Cys-Arg154Cys,Asp53Cys-Arg154Cys, Ala54Cys-Arg154Cys, Gln55Cys-Arg154Cys,Gln56Cys-Arg154Cys, Thr57Cys-Arg154Cys, Glu58Cys-Arg154Cys,Arg47Cys-Asp155Cys, Tyr48Cys-Asp155Cys, Leu49Cys-Asp155Cys,Tyr50Cys-Asp155Cys, Thr51Cys-Asp155Cys, Asp52Cys-Asp155Cys,Asp53Cys-Asp155Cys, Ala54Cys-Asp155Cys, Gln55Cys-Asp155Cys,Gln56Cys-Asp155Cys, Thr57Cys-Asp155Cys, Glu58Cys-Asp155Cys,Arg47Cys-Pro156Cys, Tyr48Cys-Pro156Cys, Leu49Cys-Pro156Cys,Tyr50Cys-Pro156Cys, Thr51Cys-Pro156Cys, Asp52Cys-Pro156Cys,Asp53Cys-Pro156Cys, Ala54Cys-Pro156Cys, Gln55Cys-Pro156Cys,Gln56Cys-Pro156Cys, Thr57Cys-Pro156Cys, Glu58Cys-Pro156Cys,Arg47Cys-Ala157Cys, Tyr48Cys-Ala157Cys, Leu49Cys-Ala157Cys,Tyr50Cys-Ala157Cys, Thr51Cys-Ala157Cys, Asp52Cys-Ala157Cys,Asp53Cys-Ala157Cys, Ala54Cys-Ala157Cys, Gln55Cys-Ala157Cys,Gln56Cys-Ala157Cys, Thr57Cys-Ala157Cys, Glu58Cys-Ala157Cys,Arg47Cys-Pro158Cys, Tyr48Cys-Pro158Cys, Leu49Cys-Pro158Cys,Tyr50Cys-Pro158Cys, Thr51Cys-Pro158Cys, Asp52Cys-Pro158Cys,Asp53Cys-Pro158Cys, Ala54Cys-Pro158Cys, Gln55Cys-Pro158Cys,Gln56Cys-Pro158Cys, Thr57Cys-Pro158Cys, Glu58Cys-Pro158Cys,Arg47Cys-Arg159Cys, Tyr48Cys-Arg159Cys, Leu49Cys-Arg159Cys,Tyr50Cys-Arg159Cys, Thr51Cys-Arg159Cys, Asp52Cys-Arg159Cys,Asp53Cys-Arg159Cys, Ala54Cys-Arg159Cys, Gln55Cys-Arg159Cys,Gln56Cys-Arg159Cys, Thr57Cys-Arg159Cys, Glu58Cys-Arg159Cys,Arg47Cys-G160Cys, Tyr48Cys-G160Cys, Leu49Cys-G160Cys,Tyr50Cys-Gly160Cys, Thr51Cys-Gly160Cys, Asp52Cys-Gly160Cys,Asp53Cys-Gly160Cys, Ala54Cys-Gly160Cys, Gln55Cys-Gly160Cys,Gln56Cys-Gly160Cys, Thr57Cys-Gly160Cys, Glu58Cys-Gly160Cys,Arg47Cys-Pro161Cys, Tyr48Cys-Pro161Cys, Leu49Cys-Pro161Cys,Tyr50Cys-Pro161Cys, Thr51Cys-Pro161Cys, Asp52Cys-Pro161Cys,Asp53Cys-Pro161Cys, Ala54Cys-Pro161Cys, Gln55Cys-Pro161Cys,Gln56Cys-Pro161Cys, Thr57Cys-Pro161Cys, Glu58Cys-Pro161Cys,Arg47Cys-Ala162Cys, Tyr48Cys-Ala162Cys, Leu49Cys-Ala162Cys,Tyr50Cys-Ala162Cys, Thr51Cys-Ala162Cys, Asp52Cys-Ala162Cys,Asp53Cys-Ala162Cys, Ala54Cys-Ala162Cys, Gln55Cys-Ala162Cys,Gln56Cys-Ala162Cys, Thr57Cys-Ala162Cys, Glu58Cys-Ala162Cys,Arg47Cys-Arg163Cys, Tyr48Cys-Arg163Cys, Leu49Cys-Arg163Cys,Tyr50Cys-Arg163Cys, Thr51Cys-Arg163Cys, Asp52Cys-Arg163Cys,Asp53Cys-Arg163Cys, Ala54Cys-Arg163Cys, Gln55Cys-Arg163Cys,Gln56Cys-Arg163Cys, Thr57Cys-Arg163Cys, Glu58Cys-Arg163Cys

Another aspect of the present invention provides fusion proteinscomprising variants of wild-type human FGF21, or a biologically activepeptide thereof, comprising a substitution of any charged and/or polarbut uncharged amino acid at any of the amino acid positions indicated inthe first embodiment of the present invention combined with thesubstitution of a cysteine at two or more amino acid positions indicatedin the second embodiment of the invention.

Improvements of the Fusion Proteins of the Invention Over Wild TypeProtein Comparators and Variants Thereof

It is well known in the art that a significant challenge in thedevelopment of protein pharmaceuticals is to deal with the physical andchemical instabilities of proteins. This is even more apparent when aprotein pharmaceutical formulation is intended to be a multiple use,injectable formulation requiring a stable, concentrated and preservedsolution, while maintaining a favorable bioactivity profile. Biophysicalcharacterization of wild-type FGF21 in the literature established that aconcentrated protein solution (>5 mg/ml), when exposed to stressconditions, such as high temperature or low pH, lead to acceleratedassociation and aggregation (i.e., poor physical stability andbiopharmaceutical properties). Exposure of a concentrated proteinsolution of FGF21 to pharmaceutical preservatives (e.g., m-cresol) alsohad a negative impact on physical stability.

Therefore, an embodiment of the present invention is to enhance physicalstability of concentrated solutions, while maintaining chemicalstability and biological potency, under both physiological and preservedformulation conditions. It is thought that association and aggregationmay result from hydrophobic interactions, since, at a given proteinconcentration, temperature, and ionic strength have considerable impacton physical stability. For the most part, non-conserved, presumedsurface exposed amino acid residues were targeted. The local environmentof these residues was analyzed and, those that were not deemedstructurally important were selected for mutagenesis. One method toinitiate specific changes is to further decrease the pl of the proteinby introducing glutamic acid residues (“glutamic acid scan”). It ishypothesized that the introduction of charged substitutes would inhibithydrophobic-mediated aggregation via charge-charge repulsion andpotentially improve preservative compatibility. In addition, one skilledin the art would also recognize that with sufficient degree ofmutagenesis the pl could be shifted into a basic pH range by theintroduction of positive charge with or without concomitant decrease innegative charge, thus allowing for charge-charge repulsion.

An additional difficulty associated with therapeutic applications ofwild-type FGF21 as a biotherapeutic, for instance, is that its half-lifeis very short in vivo (on the order of 0.5 and 2 h, respectively, inmouse and primate). There is hence a need to develop follow-up compoundsthat are more efficacious either through higher potency or longerhalf-life. The fusion proteins of the invention were developed as a wayto achieve the desirable effects of FGF21 treatment at a higher potencyand in a half-life-extended formulation.

As described further herein, the fusion proteins of the invention havehalf-lives of greater than two weeks in the mouse, compared to the muchshorter half-life of wild-type FGF21 and the 17 hour half-life of fusionprotein Fc-L (15)-FGF21 (L98R, P171G, A180E) in PCT PublicationWO10/129600. The fusion proteins of the invention also demonstrateimproved half-life and pharmacokinetic properties compared to PEGylatedV76, as described herein and in U.S. patent application 61/415,476,filed on Nov. 19, 2010.

Furthermore, the Fc-FGF21 fusion proteins of the invention at 1 mpk aremore efficacious than V76 at 5 mpk on reducing glucose, insulin, bodyweight and liver lipid. In a 12-day treatment study in ob/ob mice, thefusion proteins show the following % changes from vehicle (all of thefusions are administered at 1.0 mg/kg, and V76 is administered at 5.0mg/kg):

Total glucose (AUC) % change from vehicle: V76 is −42%; V101 is −53%,V103 is −46%, and V188 is −42%;

Total plasma insulin % change from vehicle: V76 is −46%; V101 is −82%,V103 is −69%, and V188 is −59%;

Total body weight % change from vehicle: V76 is −7%; V101 is −12%, V103is −12%, and V188 is −11%; and

Total liver lipid % change from vehicle: V76 is −30%; V101 is −44%, V103is −50%, and V188 is −51%.

Similarly, in vitro assays reveal the same 5-fold or greater potency ofthe fusion proteins of the invention over V76:

In the pERK in human adipocytes assay (mean EC50±SEM), V76 is 21±2 nM(n=3); V101 is 1.0±0.1 nM (n=3), V103 is 1.3±0.2 nM (n=3), and V188 is1.4±0.4 nM (n=3);

In the pERK in HEK293 with human βklotho assay (mean EC50±SEM), V76 is13±4 nM (n=5), V101 is 0.60±0.06 nM (n=5), V103 is 0.9±0.3 nM (n=5), andV188 is 0.4±0.1 nM (n=3); and

In the glucose uptake in mouse adipocytes assay (mean EC50±SEM), V76 is5±1 nM (n=3), V101 is 0.60±0.06 nM (n=3), V103 is 0.60±0.07 nM (n=3),and V188 is 0.48±0.14 nM (n=3).

Although the embodiments of the present invention concern the physicaland chemical stability under both physiological and preservedpharmaceutical formulation conditions, maintaining the biologicalpotency of the fusion proteins of the invention as compared to, e.g.,wild-type FGF21 is an important factor of consideration as well.Therefore, the biological potency of the proteins of the presentinvention is defined by the ability of the proteins to affect glucoseuptake and/or the lowering of plasma glucose levels, as shown herein inthe examples.

The proteins, polypeptides, and/or peptides of the inventionadministered according to this invention may be generated and/orisolated by any means known in the art. The most preferred method forproducing the variant is through recombinant DNA methodologies and iswell known to those skilled in the art. Such methods are described inCurrent Protocols in Molecular Biology (John Wiley & Sons, Inc.), whichis incorporated herein by reference.

Additionally, the preferred embodiments include a biologically activepeptide derived from the variant described herein. Such a peptide willcontain at least one of the substitutions described and the variant willpossess biological activity. The peptide may be produced by any and allmeans known to those skilled in the art, examples of which included butare not limited to enzymatic digestion, chemical synthesis orrecombinant DNA methodologies.

It is established in the art that fragments of peptides of certainfibroblast growth factors are biologically active. See for example,Baird et al., Proc. Natl. Acad. Sci (USA) 85:2324-2328 (1988), and J.Cell. Phys. Suppl. 5:101-106 (1987). Therefore, the selection offragments or peptides of the variant is based on criteria known in theart. For example, it is known that dipeptidyl peptidase IV (DPP-IV, orDPP-4) is a serine type protease involved in inactivation ofneuropeptides, endocrine peptides, and cytokines (Damme et al. Chem.Immunol. 72: 42-56, (1999)). The N-terminus of FGF21 (HisProllePro)contains two dipeptides that could potentially be substrates to DPP-IV,resulting in a fragment of FGF21 truncated at the N-terminus by 4 aminoacids. Unexpectedly, this fragment of wild-type FGF21 has beendemonstrated to retain biological activity, thus, proteins of thepresent invention truncated at the N-terminus by up to 4 amino acids, isan embodiment of the present invention.

The invention also encompasses polynucleotides encoding theabove-described variants that may be in the form of RNA or in the formof DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNAmay be double-stranded or single-stranded. The coding sequences thatencode the proteins of the present invention may vary as a result of theredundancy or degeneracy of the genetic code.

The polynucleotides that encode for the fusion proteins of the inventionmay include the following: only the coding sequence for the variant, thecoding sequence for the variant and additional coding sequence such as afunctional polypeptide, or a leader or secretory sequence or apro-protein sequence; the coding sequence for the variant and non-codingsequence, such as introns or non-coding sequence 5′ and/or 3′ of thecoding sequence for the variant. Thus the term “polynucleotide encodinga variant” encompasses a polynucleotide that may include not only codingsequence for the variant but also a polynucleotide, which includesadditional coding and/or non-coding sequence.

The invention further relates to variants of the describedpolynucleotides that encode for fragments, analogs and derivatives ofthe polypeptide that contain the indicated substitutions. The variant ofthe polynucleotide may be a naturally occurring allelic variant of thehuman FGF21 sequence, a non-naturally occurring variant, or a truncatedvariant as described above. Thus, the present invention also includespolynucleotides encoding the variants described above, as well asvariants of such polynucleotides, which variants encode for a fragment,derivative or analog of the disclosed variant. Such nucleotide variantsinclude deletion variants, substitution variants, truncated variants,and addition or insertion variants as long as at least one of theindicated amino acid substitutions of the first or second embodiments ispresent.

The polynucleotides of the invention will be expressed in hosts afterthe sequences have been operably linked to (i.e., positioned to ensurethe functioning of) an expression control sequence. These expressionvectors are typically replicable in the host organisms either asepisomes or as an integral part of the host chromosomal DNA. Commonly,expression vectors will contain selection markers, e.g., tetracycline,neomycin, and dihydrofolate reductase, to permit detection of thosecells transformed with the desired DNA sequences. The FGF21 variant canbe expressed in mammalian cells, insect, yeast, bacterial or other cellsunder the control of appropriate promoters. Cell free translationsystems can also be employed to produce such proteins using RNAs derivedfrom DNA constructs of the present invention.

E. coli is a prokaryotic host useful particularly for cloning thepolynucleotides of the present invention. Other microbial hosts suitablefor use include Bacillus subtilus, Salmonella typhimurium, and variousspecies of Serratia, Pseudomonas, Streptococcus, and Staphylococcus,although others may also be employed as a matter of choice. In theseprokaryotic hosts, one can also make expression vectors, which willtypically contain expression control sequences compatible with the hostcell (e.g., an origin of replication). In addition, any of a number ofwell-known promoters may be present, such as the lactose promotersystem, a tryptophan (Trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phages lambda or T7. The promoterswill typically control expression, optionally with an operator sequence,and have ribosome binding site sequences and the like, for initiatingand completing transcription and translation.

One skilled in the art of expression of proteins will recognize thatmethionine or methionine-arginine sequence can be introduced at theN-terminus of the mature sequence (SEQ ID NO: 3) for expression in E.coli and are contemplated within the context of this invention. Thus,unless otherwise noted, proteins of the present invention expressed inE. coli have a methionine sequence introduced at the N-terminus.

Other microbes, such as yeast or fungi, may also be used for expression.Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,and Pichia angusta are examples of preferred yeast hosts, with suitablevectors having expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences and the like as desired.Aspergillus niger, Trichoderma reesei; and Schizophyllum commune, areexamples of fungi hosts, although others may also be employed as amatter of choice.

Mammalian tissue cell culture may also be used to express and producethe polypeptides of the present invention. Eukaryotic cells are actuallypreferred, because a number of suitable host cell lines capable ofsecreting intact variants have been developed in the art, and includethe CHO cell lines, various COS cell lines, NSO cells, Syrian HamsterOvary cell lines, HeLa cells, or human embryonic kidney cell lines (i.e.HEK293, HEK293EBNA).

Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer,and necessary processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Preferred expression control sequences arepromoters derived from SV40, adenovirus, bovine papilloma virus,cytomegalovirus, Raus sarcoma virus, and the like. Preferredpolyadenylation sites include sequences derived from SV40 and bovinegrowth hormone.

The vectors containing the polynucleotide sequences of interest (e.g.,the fusion proteins of the invention and expression control sequences)can be transferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation may be used for other cellularhosts.

Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology 182: 83-9 (1990) and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, NY (1982). The purificationstep(s) selected will depend, for example, on the nature of theproduction process used for the fusion proteins of the invention.

The proteins, polypeptides, and/or peptides of the invention, e.g., thedual activity fusion proteins of the invention, should be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the patient, the site of delivery ofthe protein compositions, the method of administration, the schedulingof administration, and other factors known to practitioners. The“therapeutically effective amount” of the fusion proteins of theinvention for purposes herein is thus determined by such considerations.

The pharmaceutical compositions of the proteins of the present inventionmay be administered by any means that achieve the generally intendedpurpose: to treat type 1 and type 2 diabetes mellitus, obesity,metabolic syndrome, or critically ill patients. Non-limiting permissiblemeans of administration include, for example, by inhalation orsuppository or to mucosal tissue such as by lavage to vaginal, rectal,urethral, buccal and sublingual tissue, orally, nasally, topically,intranasally, intraperitoneally, parenterally, intravenously,intramuscularly, intrasternally, by intraarticular injection,intralymphatically, interstitially, intra-arterially, subcutaneously,intrasynovial, transepithelial, and transdermally. In some embodiments,the pharmaceutical compositions are administered by lavage, orally orinter-arterially. Other suitable methods of introduction can alsoinclude rechargeable or biodegradable devices and slow or sustainedrelease polymeric devices. The pharmaceutical compositions of thisinvention can also be administered as part of a combinatorial therapywith other known metabolic agents.

The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. Compositions withinthe scope of the invention include all compositions wherein an FGF21variant is present in an amount that is effective to achieve the desiredmedical effect for treatment type 1 or type 2 diabetes mellitus,obesity, or metabolic syndrome. While individual needs may vary from onepatient to another, the determination of the optimal ranges of effectiveamounts of all of the components is within the ability of the clinicianof ordinary skill.

The proteins of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions. A desiredformulation would be one that is a stable lyophilized product that isreconstituted with an appropriate diluent or an aqueous solution of highpurity with optional pharmaceutically acceptable carriers,preservatives, excipients or stabilizers [Remington's PharmaceuticalSciences 16th edition (1980)]. The proteins of the present invention maybe combined with a pharmaceutically acceptable buffer, and the pHadjusted to provide acceptable stability, and a pH acceptable foradministration.

For parenteral administration, in one embodiment, the fusion proteins ofthe invention are formulated generally by mixing one or more of them atthe desired degree of purity, in a unit dosage injectable form(solution, suspension, or emulsion), with a pharmaceutically acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. Preferably, one or more pharmaceutically acceptableanti-microbial agents may be added. Phenol, m-cresol, and benzyl alcoholare preferred pharmaceutically acceptable anti-microbial agents.

Optionally, one or more pharmaceutically acceptable salts may be addedto adjust the ionic strength or tonicity. One or more excipients may beadded to further adjust the isotonicity of the formulation. Glycerin,sodium chloride, and mannitol are examples of an isotonicity adjustingexcipient.

Those skilled in the art can readily optimize pharmaceutically effectivedosages and administration regimens for therapeutic compositionscomprising Proteins of the invention, as determined by good medicalpractice and the clinical condition of the individual patient. A typicaldose range for the proteins of the present invention will range fromabout 0.01 mg per day to about 1000 mg per day (or about 0.05 mg perweek to about 5000 mg per week administered once per week) for an adult.Preferably, the dosage ranges from about 0.1 mg per day to about 100 mgper day (or about 0.5 mg per week to about 500 mg per week administeredonce per week), more preferably from about 1.0 mg/day to about 10 mg/day(or about 5 mg per week to about 50 mg per week administered once perweek). Most preferably, the dosage is about 1-5 mg/day (or about 5 mgper week to about 25 mg per week administered once per week). Theappropriate dose of an FGF21 variant administered will result inlowering blood glucose levels and increasing energy expenditure byfaster and more efficient glucose utilization, and thus is useful fortreating type 1 and type 2 diabetes mellitus, obesity and metabolicsyndrome.

In addition, because hyperglycemia and insulin resistance are common incritically ill patients given nutritional support, some ICUs administerinsulin to treat excessive hyperglycemia in fed critically ill patients.In fact, recent studies document the use of exogenous insulin tomaintain blood glucose at a level no higher than 110 mg per deciliterreduced morbidity and mortality among critically ill patients in thesurgical intensive care unit, regardless of whether they had a historyof diabetes (Van den Berghe et al. N Engl J Med., 345(19):1359, (2001)).Thus, proteins of the present invention are uniquely suited to helprestore metabolic stability in metabolically unstable critically illpatients. Proteins of the invention such as those containing variants ofFGF21 are unique in that they stimulate glucose uptake and enhancesinsulin sensitivity but do not induce hypoglycemia.

In another aspect of the present invention, proteins of the inventionfor use as a medicament for the treatment of obesity, type 1 and type 2diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liverdisease (NAFLD), nonalcoholic steatohepatitis (NASH), insulinresistance, hyperinsulinemia, glucose intolerance, hyperglycemia,metabolic syndrome, acute myocardial infarction, conditions associatedwith severe inactivating mutations in the insulin receptor, and othermetabolic disorders is contemplated.

Site-Specific FGF21 Mutants

In some embodiments, the fusion proteins of the invention includeadditional FGF21 mutants or FGF21 analogues with unnatural amino acids.

In some embodiments, the fusion proteins of the invention comprise FGF21agonists with one or more of the following additional modifications ofwild-type FGF21:

(i) additional disulfides, unnatural amino acids, or modifications topromote dimerization such as formation of a disulfide at R154C orintroduction of a cysteine at another site, or dimerization through afused Fc domain, or dimer formation through a cross-linker such as abifunctional PEG;

(ii) fragments of FGF21;

(iii) proteins selected to have FGF21 activity (binding to beta-klothoand binding and activation of the FGFR's); and

(iv) an FGF21 mimetic antibody (of various formats such as Fab, unibody,svFc etc.).

In some embodiments, the fusion proteins of the invention comprise oneor more of the following linkers: a simple amide bond, short peptides(particularly Ser/Gly repeats), additional residues from the FGF21translated sequence, or a larger linker up to an entire protein (such asan Fc domain, an HSA-binding helix bundle, HSA, etc.). The two moietiescan also be linked by other chemical means, such as through unnaturalamino acids or standard chemical linkers (maleimide-Cys, NHS-Lys, click,etc.)

Other embodiments of the invention include but are not limited to thefollowing attachments, for half-life extension: HSA-binding lipid orsmall molecule or micelle to either the monomeric or a dimeric versionof the fusion.

In certain embodiments of the invention, other attachments may be madeto proteins, polypeptides, and/or peptides of the invention, to achievehalf-life extension and other improved biological properties. They caninclude attaching PEG-cholesterol conjugates (including micelles andliposomes) to the proteins, polypeptides, and/or peptides of theinvention, and/or attaching sugars (glycosylate) to the proteins,polypeptides, and/or peptides of the invention. In still otherembodiments, similar techniques are employed to add conjugates of, e.g.,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, or carbohydrate shields to proteins, polypeptides, and/orpeptides.

The HESylation technique, for example, couples branchedhydroxyethylstarch (HES) chains (60 kDa or 100 kDa, highly branchedamylopectin fragments from corn starch) to a protein, polypeptides,and/or peptides via reductive alkylation. Polsialation conjugatesproteins, polypeptides, and/or peptides of interest with polysialic acid(PSA) polymers in a manner similar to PEGylation. PSA polymers arenegatively charged, non-immunogenic polymers that occur naturally in thebody and are available in molecular weights of 10-50 kD.

In still other embodiments of the invention, other attachments ormodifications may be made to proteins, polypeptides, and/or peptides ofthe invention, to achieve half-life extension and other improvedbiological properties. These include the creation of recombinant PEG(rPEG) groups, and their attachment to the proteins, polypeptides,and/or peptides of the invention. As developed by the company Amunix,Inc. The rPEG technology is based on protein sequences with PEG-likeproperties that are genetically fused to biopharmaceuticals, avoidingthe extra chemical conjugation step. rPEGs are extended half-lifeexenatide constructs that contain a long unstructured tail ofhydrophilic amino acids, and which are capable of both increasing aprotein or peptide's serum half-life and slowing its rate of absorption,thus reducing the peak-trough ratio significantly. rPEGs have anincreased hydrodynamic radius and show an apparent molecular weight thatis about 15-fold their actual molecular weight, mimicking the wayPEGylation achieves a long serum half-life.

Truncated FGF21 Polypeptides

One embodiment of the present invention is directed to truncated formsof the mature FGF21 polypeptide (SEQ ID NO:3). This embodiment of thepresent invention arose from an effort to identify truncated FGF21polypeptides that are capable of providing an activity that is similar,and in some instances superior, to untruncated forms of the mature FGF21polypeptide.

As used herein, the term “truncated FGF21 polypeptide” refers to anFGF21 polypeptide in which amino acid residues have been removed fromthe amino-terminal (or N-terminal) end of the FGF21 polypeptide, aminoacid residues have been removed from the carboxyl-terminal (orC-terminal) end of the FGF21 polypeptide, or amino acid residues havebeen removed from both the amino-terminal and carboxyl-terminal ends ofthe FGF21 polypeptide. The various truncations disclosed herein wereprepared as described herein.

The activity of N-terminally truncated FGF21 polypeptides andC-terminally truncated FGF21 polypeptides can be assayed using an invitro phospho-ERK assay. Specific details of the in vitro assays thatcan be used to examine the activity of truncated FGF21 polypeptides canbe found in the examples.

The activity of the truncated FGF21 polypeptides of the presentinvention can also be assessed in an in vivo assay, such as ob/ob mice.Generally, to assess the in vivo activity of a truncated FGF21polypeptide, the truncated FGF21 polypeptide can be administered to atest animal intraperitoneally. After a desired incubation period (e.g.,one hour or more), a blood sample can be drawn, and blood glucose levelscan be measured.

a. N-Terminal Truncations

In some embodiments of the present invention, N-terminal truncationscomprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues from theN-terminal end of the mature FGF21 polypeptide. Truncated FGF21polypeptides having N-terminal truncations of fewer than 9 amino acidresidues retain the ability of the mature FGF21 polypeptide to lowerblood glucose in an individual. Accordingly, in particular embodiments,the present invention encompasses truncated forms of the mature FGF21polypeptide or FGF21 protein variants having N-terminal truncations of1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues.

b. C-Terminal Truncations

In some embodiments of the present invention, C-terminal truncationscomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residuesfrom the C-terminal end of the mature FGF21 polypeptide. Truncated FGF21polypeptides having C-terminal truncations of fewer than 13 amino acidresidues exhibited an efficacy of at least 50% of the efficacy ofwild-type FGF21 in an in vitro ELK-luciferase assay (Yie J. et al. FEBSLetts 583:19-24 (2009)), indicating that these FGF21 mutants retain theability of the mature FGF21 polypeptide to lower blood glucose in anindividual. Accordingly, in particular embodiments, the presentinvention encompasses truncated forms of the mature FGF21 polypeptide orFGF21 protein variants having C-terminal truncations of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 amino acid residues.

c. N-Terminal and C-Terminal Truncations

In some embodiments of the present invention, truncated FGF21polypeptides can have a combination of N-terminal and C-terminaltruncations. Truncated FGF21 polypeptides having a combination ofN-terminal and C-terminal truncations share the activity ofcorresponding truncated FGF21 polypeptides having either the N-terminalor C-terminal truncations alone. In other words, truncated FGF21polypeptides having both N-terminal truncations of fewer than 9 aminoacid residues and C-terminal truncations of fewer than 13 amino acidresidues possess similar or greater blood glucose-lowering activity astruncated FGF21 polypeptides having N-terminal truncations of fewer than9 amino acid residues or truncated FGF21 polypeptides having C-terminaltruncations of fewer than 13 amino acid residues. Accordingly, inparticular embodiments, the present invention encompasses truncatedforms of the mature FGF21 polypeptide or FGF21 protein variants havingboth N-terminal truncations of 1, 2, 3, 4, 5, 6, 7, or 8 amino acidresidues and C-terminal truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 amino acid residues.

As with all FGF21 variants of the present invention, truncated FGF21polypeptides can optionally comprise an amino-terminal methionineresidue, which can be introduced by directed mutation or as a result ofa bacterial expression process.

The truncated FGF21 polypeptides of the present invention can beprepared as described in the examples described herein. Those ofordinary skill in the art, familiar with standard molecular biologytechniques, can employ that knowledge, coupled with the instantdisclosure, to make and use the truncated FGF21 polypeptides of thepresent invention. Standard techniques can be used for recombinant DNA,oligonucleotide synthesis, tissue culture, and transformation (e.g.,electroporation, lipofection). See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, supra, which is incorporated herein byreference for any purpose. Enzymatic reactions and purificationtechniques can be performed according to manufacturer's specifications,as commonly accomplished in the art, or as described herein. Unlessspecific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsyntheses; chemical analyses; pharmaceutical preparation, formulation,and delivery; and treatment of patients.

The truncated FGF21 polypeptides of the present invention can also befused to another entity, which can impart additional properties to thetruncated FGF21 polypeptide. In one embodiment of the present invention,a truncated FGF21 polypeptide can be fused to an IgG constant domain orfragment thereof (e.g., the Fc region), Human Serum Albumin (HSA), oralbumin-binding polypeptides. Such fusion can be accomplished usingknown molecular biological methods and/or the guidance provided herein.The benefits of such fusion polypeptides, as well as methods for makingsuch fusion polypeptides, are discussed in more detail herein.

FGF21 Fusion Proteins

As used herein, the term “FGF21 fusion polypeptide” or “FGF21 fusionprotein” refers to a fusion of one or more amino acid residues (such asa heterologous protein or peptide) at the N-terminus or C-terminus ofany FGF21 protein variant described herein.

FGF21 fusion proteins can be made by fusing heterologous sequences ateither the N-terminus or at the C-terminus of, for example, an FGF21protein variant, as defined herein. As described herein, a heterologoussequence can be an amino acid sequence or a non-amino acid-containingpolymer. Heterologous sequences can be fused either directly to theFGF21 protein variant or via a linker or adapter molecule. A linker oradapter molecule can be one or more amino acid residues (or -mers),e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues (or -mers), preferably from10 to 50 amino acid residues (or -mers), e.g., 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues (or -mers), andmore preferably from 15 to 35 amino acid residues (or -mers). A linkeror adapter molecule can also be designed with a cleavage site for a DNArestriction endonuclease or for a protease to allow for the separationof the fused moieties.

Heterologous peptides and polypeptides include, but are not limited to,an epitope to allow for the detection and/or isolation of an FGF21protein variant; a transmembrane receptor protein or a portion thereof,such as an extracellular domain or a transmembrane and intracellulardomain; a ligand or a portion thereof which binds to a transmembranereceptor protein; an enzyme or portion thereof which is catalyticallyactive; a polypeptide or peptide which promotes oligomerization, such asa leucine zipper domain; a polypeptide or peptide which increasesstability, such as an immunoglobulin constant region; a functional ornon-functional antibody, or a heavy or light chain thereof; and apolypeptide which has an activity, such as a therapeutic activity,different from the FGF21 protein variants of the present invention. Alsoencompassed by the present invention are FGF21 mutants fused to humanserum albumin (HSA).

a. Fc Fusions

In one embodiment of the present invention, an FGF21 protein variant isfused to one or more domains of an Fc region of human IgG. Antibodiescomprise two functionally independent parts, a variable domain known as“Fab,” that binds an antigen, and a constant domain known as “Fc,” thatis involved in effector functions such as complement activation andattack by phagocytic cells. An Fc has a long serum half-life, whereas aFab is short-lived (Capon et al., 1989, Nature 337: 525-31). When joinedtogether with a therapeutic protein, an Fc domain can provide longerhalf-life or incorporate such functions as Fc receptor binding, proteinA binding, complement fixation, and perhaps even placental transfer(Capon et al., 1989).

Throughout the disclosure, Fc-FGF21 refers to a fusion protein in whichthe Fc sequence is fused to the N-terminus of FGF21. Similarly,throughout the disclosure, FGF21-Fc refers to a fusion protein in whichthe Fc sequence is fused to the C-terminus of FGF21.

Preferred embodiments of the invention are Fc-FGF21 fusion proteinscomprising FGF21 variants as defined herein. Particularly preferredembodiments are Fc-FGF21 fusion proteins comprising a modified Fcfragment (e.g., an FcLALA) and FGF21 variants as defined herein.

Fusion protein can be purified, for example, by the use of a Protein Aaffinity column. Peptides and proteins fused to an Fc region have beenfound to exhibit a substantially greater half-life in vivo than theunfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regioncan be a naturally occurring Fc region, or can be altered to improvecertain qualities, such as therapeutic qualities, circulation time, orreduced aggregation.

Useful modifications of protein therapeutic agents by fusion with the“Fc” domain of an antibody are discussed in detail in PCT PublicationNo. WO 00/024782. This document discusses linkage to a “vehicle” such aspolyethylene glycol (PEG), dextran, or an Fc region.

b. Fusion Protein Linkers

When forming the fusion proteins of the present invention, a linker can,but need not, be employed. When present, the linker's chemical structuremay not critical, since it serves primarily as a spacer. The linker canbe made up of amino acids linked together by peptide bonds. In someembodiments of the present invention, the linker is made up of from 1 to20 amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. In variousembodiments, the 1 to 20 amino acids are selected from the amino acidsglycine, serine, alanine, proline, asparagine, glutamine, and lysine. Insome embodiments, a linker is made up of a majority of amino acids thatare sterically unhindered, such as glycine and alanine. In someembodiments, linkers are polyglycines, polyalanines, combinations ofglycine and alanine (such as poly(Gly-Ala)), or combinations of glycineand serine (such as poly(Gly-Ser)). While a linker of 15 amino acidresidues has been found to work particularly well for FGF21 fusionproteins, the present invention contemplates linkers of any length orcomposition.

The linkers described herein are exemplary, and linkers that are muchlonger and which include other residues are contemplated by the presentinvention. Non-peptide linkers are also contemplated by the presentinvention. For example, alkyl linkers such as can be used. These alkyllinkers can further be substituted by any non-sterically hinderinggroup, including, but not limited to, a lower alkyl (e.g., C1-C6), loweracyl, halogen (e.g., Cl, Br), CN, NH2, or phenyl. An exemplarynon-peptide linker is a polyethylene glycol linker, wherein the linkerhas a molecular weight of 100 to 5000 kD, for example, 100 to 500 kD.

Chemically-Modified Fusion Proteins

Chemically modified forms of the fusion proteins described herein,including, e.g., truncated and variant forms of the FGF21 fusionsdescribed herein, can be prepared by one skilled in the art, given thedisclosures described herein. Such chemically modified Fusion Proteinsare altered such that the chemically modified mutant is different fromthe unmodified mutant, either in the type or location of the moleculesnaturally attached to the mutant. Chemically modified mutants caninclude molecules formed by the deletion of one or morenaturally-attached chemical groups.

In one embodiment, proteins of the present invention can be modified bythe covalent attachment of one or more polymers. For example, thepolymer selected is typically water-soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. Included within the scope of suitablepolymers is a mixture of polymers. Preferably, for therapeutic use ofthe end-product preparation, the polymer will be pharmaceuticallyacceptable. Non-water soluble polymers conjugated to proteins of thepresent invention also form an aspect of the invention.

Exemplary polymers each can be of any molecular weight and can bebranched or unbranched. The polymers each typically have an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water-soluble polymer, somemolecules will weigh more and some less than the stated molecularweight). The average molecular weight of each polymer is preferablybetween about 5 kDa and about 50 kDa, more preferably between about 12kDa and about 40 kDa, and most preferably between about 20 kDa and about35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C1-C10), alkoxy-, oraryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran(such as low molecular weight dextran of, for example, about 6 kD),cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules that can be used toprepare covalently attached FGF21 protein variant multimers. Alsoencompassed by the present invention are FGF21 mutants covalentlyattached to polysialic acid.

Polysaccharide polymers are another type of water-soluble polymer thatcan be used for protein modification. Therefore, the fusion proteins ofthe invention fused to a polysaccharide polymer form embodiments of thepresent invention. Dextrans are polysaccharide polymers comprised ofindividual subunits of glucose predominantly linked by alpha 1-6linkages. The dextran itself is available in many molecular weightranges, and is readily available in molecular weights from about 1 kD toabout 70 kD. Dextran is a suitable water-soluble polymer for use as avehicle by itself or in combination with another vehicle (e.g., Fc).See, e.g., International Publication No. WO 96/11953. The use of dextranconjugated to therapeutic or diagnostic immunoglobulins has beenreported. See, e.g., European Patent Publication No. 0 315 456, which ishereby incorporated by reference. The present invention also encompassesthe use of dextran of about 1 kD to about 20 kD.

In general, chemical modification can be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemically modified polypeptides will generallycomprise the steps of: (a) reacting the polypeptide with the activatedpolymer molecule (such as a reactive ester or aldehyde derivative of thepolymer molecule) under conditions whereby a FGF21 protein variantbecomes attached to one or more polymer molecules, and (b) obtaining thereaction products. The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules to protein, the greater thepercentage of attached polymer molecule. In one embodiment of thepresent invention, chemically modified FGF21 mutants can have a singlepolymer molecule moiety at the amino-terminus (see, e.g., U.S. Pat. No.5,234,784)

In another embodiment of the present invention, Proteins of theinvention can be chemically coupled to biotin. The biotin/Proteins ofthe invention are then allowed to bind to avidin, resulting intetravalent avidin/biotin/Proteins of the invention. Proteins of theinvention can also be covalently coupled to dinitrophenol (DNP) ortrinitrophenol (TNP) and the resulting conjugates precipitated withanti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of10.

Generally, conditions that can be alleviated or modulated by theadministration of the present chemically modified FGF21 mutants includethose described herein for Proteins of the invention. However, thechemically modified FGF21 mutants disclosed herein can have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto unmodified FGF21 mutants.

Therapeutic Compositions of Fusion Proteins and Administration Thereof

The present invention also provides therapeutic compositions comprisingone or more of the fusion proteins of the invention described herein andin admixture with a pharmaceutically or physiologically acceptableformulation agent or pharmaceutically acceptable carrier selected forsuitability with the mode of administration. The compositions arespecifically contemplated in light of, e.g., the identification offusions proteins exhibiting enhanced properties.

In some embodiments the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable salts can also be present in the pharmaceutical composition,e.g., mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients is available inRemington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro,Lippincott, Williams, & Wilkins.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition can contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides; preferably sodium or potassium chloride; ormannitol sorbitol), delivery vehicles, diluents, excipients and/orpharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences(18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990), andsubsequent editions of the same, incorporated herein by reference forany purpose).

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage (see, e.g.,Remington's Pharmaceutical Sciences, supra). Such compositions caninfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the fusion protein of the invention.

The primary vehicle or carrier in a pharmaceutical composition can beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection can be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichcan further include sorbitol or a suitable substitute. In one embodimentof the present invention, dual function pharmaceutical compositions canbe prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the dual function protein product can beformulated as a lyophilizate using appropriate excipients such assucrose.

The pharmaceutical compositions containing the fusion proteins of theinvention can be selected for parenteral delivery. Alternatively, thecompositions can be selected for inhalation or for delivery through thedigestive tract, such as orally. The preparation of suchpharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention can be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired dual function protein in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is steriledistilled water in which a dual function protein is formulated as asterile, isotonic solution, properly preserved. Yet another preparationcan involve the formulation of the desired molecule with an agent, suchas injectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads, or liposomes,that provides for the controlled or sustained release of the productwhich can then be delivered via a depot injection. Hyaluronic acid canalso be used, and this can have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

In one embodiment, a pharmaceutical composition can be formulated forinhalation. For example, a dual function protein of the invention can beformulated as a dry powder for inhalation. Dual function proteininhalation solutions can also be formulated with a propellant foraerosol delivery. In yet another embodiment, solutions can be nebulized.Pulmonary administration is further described in InternationalPublication No. WO 94/20069, which describes the pulmonary delivery ofchemically modified proteins.

It is also contemplated that certain formulations can be administeredorally. In one embodiment of the present invention, Fusion Proteins ofthe invention that are administered in this fashion can be formulatedwith or without those carriers customarily used in the compounding ofsolid dosage forms such as tablets and capsules. For example, a capsulecan be designed to release the active portion of the formulation at thepoint in the gastrointestinal tract when bioavailability is maximizedand pre-systemic degradation is minimized. Additional agents can beincluded to facilitate absorption of the fusion proteins of theinvention. Diluents, flavorings, low melting point waxes, vegetableoils, lubricants, suspending agents, tablet disintegrating agents, andbinders can also be employed.

Another pharmaceutical composition can involve an effective quantity ofthe fusion proteins of the invention in a mixture with non-toxicexcipients that are suitable for the manufacture of tablets. Bydissolving the tablets in sterile water, or another appropriate vehicle,solutions can be prepared in unit-dose form. Suitable excipientsinclude, but are not limited to, inert diluents, such as calciumcarbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions comprising Fusion Proteins of theinvention will be evident to those skilled in the art, includingformulations involving Fusion Proteins of the invention in sustained- orcontrolled-delivery formulations. Techniques for formulating a varietyof other sustained- or controlled-delivery means, such as liposomecarriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art (see, e.g.,International Publication No. WO 93/15722, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. JPharm. 364: 298-327, and Freiberg & Zhu, 2004, Int. J Pharm. 282: 1-18,which discuss microsphere/microparticle preparation and use).

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 0 058 481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105),ethylene vinyl acetate (Langer et al., supra) or poly-D-3-hydroxybutyricacid (European Patent No. 0 133 988). Sustained-release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Epstein et al., 1985, Proc. Natl. Acad.Sci. U.S.A. 82: 3688-92; and European Patent Nos. 0 036 676, 0 088 046,and 0 143 949.

The pharmaceutical compositions of the invention to be used for in vivoadministration typically must be sterile. This can be accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using this method can be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration can be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits can each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

Dosages of Fusion Proteins and Administration Thereof

The effective amount of an pharmaceutical composition of the inventionto be employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the fusion protein variant is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraarterial, intraportal, orintralesional routes; by sustained release systems (which may also beinjected); or by implantation devices. Where desired, the compositionscan be administered by bolus injection or continuously by infusion, orby implantation device.

Alternatively or additionally, the composition can be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial onto which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

Therapeutic Uses of Fusion Proteins

Proteins of the invention can be used to treat, diagnose, ameliorate, orprevent a number of diseases, disorders, or conditions, including, butnot limited to metabolic disorders. In one embodiment, the metabolicdisorder to be treated is diabetes, e.g., type 2 diabetes mellitus. Inanother embodiment, the metabolic disorder is obesity. Other embodimentsinclude metabolic conditions or disorders such as type 1 diabetesmellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, metabolicsyndrome, hypertension, cardiovascular disease, acute myocardialinfarction, atherosclerosis, peripheral arterial disease, stroke, heartfailure, coronary heart disease, kidney disease, diabetic complications,neuropathy, disorders associated with severe inactivating mutations inthe insulin receptor, gastroparesis and other metabolic disorders.

In application, a disorder or condition such as type 1 or type 2diabetes mellitus or obesity can be treated by administering an FGF21protein variant as described herein to a patient in need thereof in theamount of a therapeutically effective dose. The administration can beperformed as described herein, such as by IV injection, intraperitonealinjection, intramuscular injection, or orally in the form of a tablet orliquid formation. In most situations, a desired dosage can be determinedby a clinician, as described herein, and can represent a therapeuticallyeffective dose of the FGF21 mutant polypeptide. It will be apparent tothose of skill in the art that a therapeutically effective dose of FGF21mutant polypeptide will depend, inter alia, upon the administrationschedule, the unit dose of antigen administered, whether the nucleicacid molecule or polypeptide is administered in combination with othertherapeutic agents, the immune status and the health of the recipient.The term “therapeutically effective dose,” as used herein, means thatamount of FGF21 mutant polypeptide that elicits the biological ormedicinal response in a tissue system, animal, or human being sought bya researcher, medical doctor, or other clinician, which includesalleviation of the symptoms of the disease or disorder being treated.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); and Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989).

EXAMPLES Example 1: Preparation of FGF21 Variant Proteins

Expression Construct for FGF21 V76:

The FGF21 variants were cloned into the modified E. coli expressionvector pET30a, described by Achmuller et al. (2007) (Nature Methods4:1037-1043), to generate in-frame fusions to a hexa-histidine tagfollowed by the N^(pro)-EDDIE tag at the N-terminus of FGF21 (aa33-209).

Expression and Purification of FGF21 V76:

The pET30a-His-N^(pro)-EDDIE-FGF21 expression plasmid was transformedinto E. coli BL21 Star (DE3) competent cells (Invitrogen). Overnightgrowth from a single colony of freshly transformed cells was carried outin 50 mL of Terrific Broth (TB) containing 50 μg/mL of kanamycin at 37°C. The pre-culture was transferred into 1 L of TB medium with kanamycinand cultured in baffled flasks at 37° C. with shaking at 250 rpm. After6 hour of culture, expression of FGF21 was induced by the addition ofIPTG at a final concentration of 1 mM, and the cultures were grownovernight at 37° C. The cells were then harvested and resuspended into50 mL of ice-cold lysis buffer; 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mMEDTA, followed by lysis using a Microfluidizer™.

Inclusion bodies (IBs) were precipitated by centrifugation at 30,000×gfor 1 hour at 4° C. The IBs were washed with 50 mM Tris-HCl, pH 8, 150mM NaCl and then dissolved into 30 mL of dissolving buffer; 10 mMTris-HCl, pH 8, 100 mM NaH₂PO₄, 6 M GnHCl. The dissolved IBs wereclarified by centrifugation at 30,000×g for 1 hour at 25° C. The IBsolution was loaded onto a 5 mL column of Ni-NTA high performance resin(GE Healthcare) equilibrated with the dissolving buffer. Proteins boundto the resin were eluted by decreasing the pH to 4.5. The eluate wasconditioned by adjusting pH and adding dithiothreitol (DTT) at aconcentration of 20 mM. The conditioned eluate was slowly diluted into 1L of refolding buffer; 50 mM Tris-HCl, pH 8, 0.5 M arginine, 20 mM DTT,followed by incubation for 2 days at 4° C. The diluted sample wasconcentrated and buffer-exchanged into 20 mM Tris-HCl, pH 9 using anultrafiltration method. The concentrated sample was loaded onto a 10 mLcolumn of Q sepharose fast flow resin (GE Healthcare) equilibrated with20 mM Tri-HCl (pH 9).

After washing the resin with the equilibration buffer, proteins bound tothe resin were eluted with 20 mM Tris-HCl, pH 9, 500 mM NaCl. To removethe cleaved off His-N^(pro) fusion fragment and any uncleaved fusionprotein from the refolded FGF21 protein, the eluate was loaded onto a 5mL column of Ni-NTA high performance resin equilibrated with 20 mM Tris,pH 8.0, 50 mM imidazole, and the flow-through fraction containing FGF21was collected. To reduce endotoxin levels, the FGF21 fraction wastreated with an EndoTrap HD resin (Hyglos) equilibrated with 10 mM Tris,pH 8, 50 mM imidazole, 500 mM NaCl, 1 mM CaCl₂. The low-endotoxin samplewas dialyzed against PBS and then sterilized with a 0.22 μm filter. Thepurified FGF21 protein was snap-frozen in liquid nitrogen and stored at−80° C. Protein concentration was determined by absorbance at 280 nmusing 9362 M-1 cm-1 as the molar extinction coefficient for FGF21.Protein purity and integrity were determined by HPLC, SDS-PAGE andliquid chromatography-mass spectrometry.

Cysteine PEGylation of FGF21 Variants:

FGF21 Variant V76 (R154C) variant has the tendency to dimerize via theengineered cysteine; therefore, prior to PEGylation the protein solution(typically 5 mg/mL in Tris buffer) was mildly reduced with 5 mMmercaptoethylamine for 30 minutes on ice and immediately desalted in 20mM Tris, pH 7. The freshly reduced protein (typically 3 mg/mL) was thenimmediately PEGylated with 1.5 equivalent of 40 kDa branchedmaleimido-PEG reagent (NOF, Cat. #GL2-400MA from the Sunbright series)for 3 hours on ice. The PEGylated protein was finally purified by anionexchange chromatography (MonoQ) with overall yields of about 25%.

Expression Constructs for Fc-FGF21 Fusion Variants:

The cDNAs for human FGF21 variants encoding amino acids 33-209 werecloned into a mammalian expression vector downstream of thecytomegalovirus (CMV) promoter in-frame with N-terminal sequencesincluding a leader peptide (immunoglobulin kappa-chain) to directsecretion of the proteins, followed by an Fc domain and a short linker.

Expression and Purification of Fc-FGF21 Variants:

The Fc-FGF21 variant proteins were expressed into HEK293T cells(American Type Culture Collection). Cells were grown in suspensionculture at 37° C., 8% CO₂, in Freestyle 293 Expression Medium(Invitrogen, Cat. #12338-018) until day of transfection. Cells werecentrifuged at 1000×g for 7 min in a swinging bucket rotor and countedusing an automated cell counter. Cells were diluted in 900 mL ofFreestyle 293 media to a final concentration of 1.4×10⁶ cells/mL andplaced into a 3 L non-baffled flask (Corning, Cat. #431252). Cells weretransfected using a mixture of polyethyleneimine (PEI) and plasmid asfollows. Three mL of a sterile 1 mg/mL stock of linear, M.W. 25,000, PEI(Alfa Aesar, Cat. #43896) was added to 50 mL of Freestyle 293 media,mixed gently and incubated at 25° C. for 5 minutes. At the same time, 1mg of endotoxin-free plasmid was added to 50 mL Freestyle 293 media andsterile filtered using a 0.22 uM filter. The PEI mixture was then addedto the sterile filtered DNA, mixed gently and allowed to incubate at 25°C. for 10 minutes. The PEI-plasmid mixture was then added to the 3 Lflask containing the diluted HEK 293T cells and placed at in a shakingincubator at 125 RPM, 37° C., 8% CO₂.

On day 6 post-transfection, the cells were centrifuged at 2000×g for 10minutes and the supernatant was harvested. The supernatant was furtherclarified by filtration through a 0.8/0.2 uM filter (Pall Corporation,Cat. #4628).

Batch purification of the FGF21 protein was done by adding 1 mL ofrecombinant Protein A Sepharose Fast Flow (GE, Cat. #17-5138-03), per 20mg of expected protein to be purified, directly to the clarifiedsupernatant and incubating for 1 hour at 4° C. with gentle rotation. Thesupernatant mixture was then poured over a disposable Poly-PrepChromatography Column (Bio-Rad, Cat. #731-1550) and the flow through wasdiscarded. The retained beads were washed with 5 column volumes of DPBS,pH 7.4 (Invitrogen, Cat. #14190-144). Elution of the protein from theProtein A beads was done by adding 20 column volumes of 50 mM SodiumCitrate buffer, pH 3.0.

The elution buffer was neutralized by the addition of 20% Tris-HCLbuffer, pH 9.0. Size exclusion chromatography was preformed as asecondary polishing step by running the Protein A batch purifiedmaterial over a High Load 26/600 Superdex 200 pg column (GE, Cat.#28-9893-36). The purified protein yield was quantified by A280.SDS-Page was run to verify purity and molecular weight. Endotoxin levelwas quantified by using the Endosafe PTS system (Charles River Labs).

Example 2: Measuring FGF21 Dependent 2-Deoxyglucose (2-DOG) Uptake

FGF21 has been shown to stimulate glucose-uptake in mouse 3T3-L1adipocytes in the presence and absence of insulin, and to decrease fedand fasting blood glucose, triglycerides, and glucagon levels in ob/oband db/db mice and 8 week old ZDF rats in a dose-dependent manner, thus,providing the basis for the use of FGF21 as a therapy for treatingdiabetes and obesity (see, e.g., patent publication WO03/011213, andKharitonenkov et al., (2005) Jour. of Clinical Invest. 115:1627-1635).Also, FGF21 was observed to stimulate tyrosine phosphorylation of FGFR-1and FGFR-2 in 3T3-L1 adipocytes.

3T3-L1 fibroblasts were purchased from ATCC (Cat. # CL173). The cellswere grown to confluency in 150 cm petri-dish and were maintained inDMEM with high glucose (Invitrogen, Cat. #11995065) supplemented with10% Fetal Bovine Serum and 1% penicillin-streptomycin for an additional4 days. Cells were then differentiated in the above media supplementedwith 4 μg/mL insulin (Sigma, Cat. #I-5500), 115 μg/mL IBMX (Sigma, Cat.#I5879) and 0.0975 μg/mL dexamethasone (Sigma, Cat. #D1756) for 3 daysafter which the differentiation media was replaced with complete DMEM.One plate of differentiated 3T3-L1 adipocytes were seeded into four96-well plates the day after medium replacement.

The adipocytes were then treated with FGF21-WT and FGF21 variant protein(see Table 2 for list of variants; 30 pM to 100 nM is the typicalconcentration range used) overnight in complete medium. The adipocytestreated with FGF21 samples were serum starved in 50 μL per well KRHbuffer (0.75% NaCl; 0.038% KCl; 0.0196% CaCl₂; 0.032% MgSO₄; 0.025MHEPES, pH 7.5; 0.5% BSA; 2 mM sodium pyruvate) for 2 hours. The wellsfor blank were added with 1 μL (final concentration 5 μg/ml)cytochalasin B for 15 min. [3H]-2-DOG (20.6 mCi/mmoL, 1 mCi/mL) wasdiluted 1:20 in 5.1 mM cold 2-DOG and 1 μL diluted 2-DOG was added perwell and the cells were incubated for 5 min. The cells were washed with100 μL/well KRH buffer three times. 40 μL/well 1% SDS was added to cellsand the cells were shaken for at least 10 minutes. 200 μL/wellscintillation fluid was added and the plates were shaken overnight andread in beta-microplate reader. The values obtained from an entirecolumn/row, which were treated with cytochalasin B, was averaged andsubtracted from all other values. The data were analyzed by GraphPadPrism software, the results of which are summarized in Table 2. Fc-FGF21Fusion Variants V101, V103 and V188 are superior to PEGylated FGF21Variant V76 in for induction of 2-deoxyglucose uptake by mouse 3T3L1adipocytes.

Example 3: pERK in Cell Western (ICW) Assay

HEK293 cells stably transfected with human β-klotho were cultured inDMEM high glucose, 10% FBS, 1% PS and 600 ng/mL G418 are seeded inpoly-D-lysine coated 96-well plates (BD bioscience, Cat. #356640) at30,000 cells per well overnight. The cells were serum starved in DMEMhigh glucose, 0.5% BSA and 10 mM HEPES for 4 hours. WT FGF21 and theFGF21 variants (see Table 3 for list of variants) were diluted tovarious concentrations (100 pM to 300 nM is the typical concentrationrange used) in starvation medium. The cells were stimulated with FGF21for 10 minutes. Following FGF21 or FGF21 Variant protein stimulation,the media was aspirated from the wells and the cells were washed oncewith 100 μL cold PBS and then fixed with 100 μl of 4% formaldehyde for15 minutes at room temperature and followed by an additional 10 minuteincubation with 100 μL ice-cold methanol.

After fixation, the cells were washed with 0.3% Triton X-100 in PBS fourtimes, 5 minutes each. 150 μL Odyssey Blocking Buffer was added to thepermeabilized cells at room temperature for 1.5 hours. Phospho-ERK(pERK) antibody was diluted to a concentration of 0.17 μg/mL (1:200dilution, or the dilutions indicated), and total-ERK (tERK) antibody wasdiluted to a concentration of 2.2 μg/mL (1:200 dilution, or thedilutions indicated) in Odyssey Blocking Buffer. 50 μL was added toevery well, omitting one column which was only treated with secondaryantibody to normalize for background. The plate was covered with the wetpaper tower and lid to prevent evaporation and then incubated at 4° C.overnight.

Afterwards, the primary antibody was aspirated and the cells were washedfour times with 0.3% Tween 20 in PBS for 5 minutes each. During thewashing, the secondary antibody reaction mixture was prepared in OdysseyBlocking Buffer containing 1:1000-diluted (or the dilutions indicated)goat anti-mouse Alexa 680 and 1:1000-diluted (or the dilutionsindicated) IRDye800 goat anti-rabbit antibody. Once the washing wascompleted, 40 μL of the reaction mixture was added to each well. Plateswere covered with black lid to protect the secondary antibody fromlight, and plates were incubated at room temperature for 1 hour on ashaker. Finally, the cells were washed again four times with 0.3% Tween20 in PBS for 5 minutes each and then scanned on the LI-COR BioscienceOdyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebr.) inthe 700 nm (red) and 800 nm (green) channels. Alexa 680 stained the tERKwith far-red fluorescence (emission wavelength 668 nm), while IRDye800stained the pERK with green fluorescence (emission wavelength 800 nm).To eliminate the fluorescent background, the values obtained from anentire column/row, which was treated with only secondary antibody, wasaveraged and subtracted from all other values obtained from the plate.For normalization of the amount of pERK present in each sample, thevalues for pERK in each well was divided by the values of tERK. The datawere analyzed by GraphPad Prism software, the results of which aresummarized in Table 2. Fc-FGF21 Fusion Variants V101, V103 and V188 aresuperior to PEGylated FGF21 Variant V76 in this ERK phosphorylationassay.

TABLE 2 Summary of ERK in cell Western and Mouse 3T3L1 Adipocyte GlucoseUptake Assay Results pERK Glucose Uptake FGF21 (HEK293/human β-klotho)(Mouse 3T3L1 adipocytes) Variant ID EC50 ± SEM EC50 ± SEM V76   13 ± 4nM (n = 5)    5 ± 1 nM (n = 3) V101 0.60 ± 0.06 nM (n = 5)  0.60 ± 0.06nM (n = 3) V103 0.9 ± 0.3 nM (n = 5) 0.60 ± 0.07 nM (n = 3) V188 0.4 ±0.1 nM (n = 3) 0.48 ± 0.14 nM (n = 3)

Example 4: In Vivo Tests of FGF21 Variants

The ob/ob mouse is a mouse model for type 2 diabetes. The mice lackfunctional leptin and are characterized by hyperglycemia, insulinresistance, hyperphagia, hepatic steatosis and obesity. Male ob/ob mice(10-13 weeks old) were used to measure the effect on blood glucose ofthe following PEGylated FGF21 variant V76 and Fc-FGF21 fusion variantsV101, V103 and V188.

FGF21 variants or PBS vehicle were administered s.c. at 1 mg/kg (V101,V103 and V188) or s.c at 5 mg/kg V76 twice per week 12 days (4 dosestotal). On the first day of the study, tail blood glucose and bodyweight were measured and mice were allocated into different groups (n=8per group) with mean glucose and body weight matched among the groups.Blood glucose was measured using a glucometer (OneTouch). Plasma insulinwas measured on day 1 before dosing and on day 12, 24 hours post thelast dose. The results of these studies are summarized in Table 5.

The results of these studies are summarized in Table 3 and FIGS. 1-3.Fc-FGF21 Fusion Variants V101, V103 and V188 are superior to PEGylatedFGF21 Variant V76 on every endpoint measured in these studies and at afive-fold lower dose.

TABLE 3 % changes versus vehicle in plasma glucose, insulin, body weight(BW) gain, liver TG/lipid by FGF21 variants during 12-day studies inob/ob mice. Summary of 12-day treatment study in diabetic ob/ob mice (%change from vehicle) Total FGF21 Dose Glucose Plasma Body Variant ID(mg/kg) (AUC) Insulin Weight Liver lipid V76  5.0 −42% −46% −7% −30%V101 1.0 −53% −82% −12% −44% V103 1.0 −46% −69% −12% −50% V188 1.0 −42%−59% −11% −51%

Example 5: Pharmacokinetics of FGF21 Fusion Variants in Mice

To determine the pharmacokinetic profile of Fc-FGF21 Fusion VariantsV101, V103 and V188, C57BL/6J mice were injected IV with 1 mg/kg testarticle and bled at various time points out to 16 days (384 hours).Blood samples were collected into EDTA-coated microtainer tubes fromeither the submandibular or retro-orbital plexus. Approximately 50 μL ofblood was collected at each time point, yielding ˜25 μL of plasma.

To measure plasma concentrations of test articles by ELISA, 384-wellplates were coated overnight at room temperature (RT) with 2 μg/mL ofanti-Human Fc-gamma goat polyclonal antibody (30 μL/well) and thenblocked with a casein-based diluent for 2 hour at RT (100 μL/well).Diluted samples, standards, and controls were added to the plate (30μL/well) and incubated for 2 hour at RT. After the samples were removed,the wells were washed 3 times with a phosphate-based wash solution (100μL/well). The detection antibody, an HRP-labeled version of the captureantibody, was added to the plate and incubated for 1 hour at RT (30μL/well). After the plate was again washed 3 times with aphosphate-based wash solution (100 μL/well), a chemiluminescentsubstrate was added (30 μL/well) and the plate luminescence was readwithin 5 minutes using an appropriate plate reader. As shown in FIGS. 4Aand 4B the Fc-FGF21 fusion variants had a greatly extended plasmahalf-life relative to known Fc-FGF21 fusions in the art (FIG. 4A) andrelative to PEGylated FGF21 variant V76 (FIG. 4B).

Serum levels of Fc-FGF21 test articles were validated by Western blotfor comparison to levels measured by ELISA to ensure that full lengthFc-FGF21 variant and not Fc alone was being detected in the ELISA. TwouL of mouse serum was combined with 2.5 uL of 4× loading buffer, 1 uL of10× denaturant and 4 uL of dH₂O, heated to 95° C. for 5 minutes andloaded onto a 4-12% gradient polyacrylamide gel and electrophoresed for1 hour at 100 Volts (constant voltage). Samples were transferred tonitrocellulose filter paper by Western blot using the iblot system(Invitrogen, Cat. #IB1001, 7 minute run time). The nitrocellulosefilters were blocked with 30 mL of Rockland blocking solution (Cat.#MB-070), probed following the snap iblot system protocol with a goatanti-FGF21 primary antibody at a 1:2000 dilution (R&D systems, Cat.#BAF2539) and fluorescently labeled streptavidin as a secondary at a1:10000 dilution (Licor, Cat. #926-68031). Protein levels were imaged onthe Licor Odyssey system at 700 nm and compared with 2 nM control V101run on the same gel. As shown in FIG. 4C full-length Fc-FGF21 variantsV101, V103 and V188 are detectable using on a Western Blot usinganti-FGF21 antibody out to 15 days from mouse serum from thepharmacokinetic study.

Example 6: Fc-FGF21 Fusion Variants V101, V103 and V188 are ExtremelyThermodynamically Stabile

Proteins can be unfolded at specific temperature range. The temperatureof protein unfolding is an intrinsic parameter to describe thermalstability of proteins. Differential Scanning calorimetry (DSC) is usedto detect the unfolding temperature of protein. This characteristictemperature is described as melting temperature (Tm), which is the peaktemperature during protein unfolding.

Original protein samples are diluted in PBS to a concentration of ˜1mg/ml (0.5 mg/ml to 1.2 mg/ml) for a total volume of 0.5 ml. An aliquotof 0.4 ml per well diluted protein sample, standard, PBS, and DI waterare added to DSC 96-well plate. The plate is then covered by a seal.Samples were analyzed in a 96 well Differential Scanning calorimeterfrom MicroCal. The temperature was scanned from 10-110 degrees C. at arate of 1 degree per minute.

As shown in FIG. 4 D the melting temperatures of FGF21 variants V101,V103 and V188 are extremely high. This is in contrast to the lowermelting temperatures of FGF21 variant V76 and wild-type FGF21 (notshown). We attribute the improved stability of V101, V103 and V188 tothe specific addition of a second disulfide bond from the novel Q55C andG148C mutations. This type of thermodynamic stability is known toprotect proteins from proteolysis and can in addition translate intosignificantly prolonged stability in vivo and the improvedpharmacokinetic profiles exemplified by the data in FIGS. 4B and 4C.

What is claimed is:
 1. A nucleic acid coding for a fusion proteincomprising a fibroblast growth factor 21 (FGF21) variant and an Fcregion, wherein the FGF21 variant comprises an amino acid sequence withat least 95% identity to the full length hFGF21 sequence SEQ ID NO:1,and comprises at least the following mutations relative to SEQ ID NO:1:Q55C, G148C, K150R, P158S, S195A, P199G, and G202A, and one of R105K andA109T.
 2. A vector comprising the nucleic acid according to claim
 1. 3.A host cell comprising the vector of claim
 2. 4. A method of producing afusion protein, wherein the method comprises culturing the host cell ofclaim
 3. 5. The nucleic acid of claim 1, wherein the nucleic acid codesfor a fusion protein comprising the amino acid sequence of SEQ ID NO:10, 11, 12, or
 13. 6. A vector comprising the nucleic acid according toclaim
 5. 7. A host cell comprising the vector of claim
 6. 8. A method ofproducing a fusion protein, wherein the method comprises culturing thehost cell of claim
 7. 9. The nucleic acid of claim 1, wherein thenucleic acid codes for a fusion protein comprising the amino acidsequence of SEQ ID NO:
 11. 10. A vector comprising the nucleic acidaccording to claim
 9. 11. A host cell comprising the vector of claim 10.12. A method of producing a fusion protein, wherein the method comprisesculturing the host cell of claim 11.